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Abstract:

In certain aspects, the present invention provides methods for dosing a
patient with an activin-ActRIIa antagonist and methods for managing
patients treated with an activin-ActRIIa anatagonist. In certain aspects,
the methods involve measuring one or more hematologic parameters in a
patient.

Claims:

1. A method for managing a patient that has been treated with, or is a
candidate to be treated with, an activin-ActRIIa antagonist, the method
comprising monitoring in the patient one or more hematologic parameters
that correlate with increased red blood cell levels.

2. The method of claim 1, wherein the hematologic parameters are one or
more of the following: red blood cell levels, blood pressure, or iron
stores.

3. The method of claim 1, the method further comprising administering to
the patient the activin-ActRIIa antagonist in an amount that is
appropriate to the observed level of said hematologic parameter in the
patient.

4. The method of claim 1, wherein, if the patient has blood pressure
elevated above baseline or is hypertensive, dosing with the
activin-ActRIIa antagonist is reduced, delayed or terminated.

5. The method of claim 1, wherein, if the patient has blood pressure
elevated above baseline or is hypertensive, the patient is treated with a
blood pressure lowering agent prior to administration of the
activin-ActRIIa antagonist.

6. The method of claim 1, wherein if the patient has uncontrolled
hypertension, dosing with the activin-ActRIIa antagonist is reduced,
delayed or terminated.

7. The method of claim 1, wherein if the patient has a red blood cell
level greater than the normal range for patients of similar age and sex,
dosing with the activin-ActRIIa antagonist is reduced, delayed or
terminated.

8. The method of claim 1, wherein if the patient has a hemoglobin level of
greater than 15 g/dl, dosing with the activin-ActRIIa antagonist is
reduced, delayed or terminated.

9. The method of claim 1, wherein if the patient has a hemoglobin level
greater than 10, 11 or 12 g/dl, dosing with the activin-ActRIIa
antagonist is reduced, delayed or terminated.

10. The method of claim 1, wherein if the patient has iron stores that are
lower than the normal range for patients of similar age and sex, dosing
with the activin-ActRIIa antagonist is reduced, delayed or terminated.

11. The method of claim 1, wherein if the patient has a transferrin
saturation of less than 20%, dosing with the activin-ActRIIa antagonist
is reduced, delayed or terminated.

12. The method of claim 1, wherein if the patient has a ferritin level of
less than 100 ng/ml, dosing with the activin-ActRIIa antagonist is
reduced, delayed or terminated.

13. The method of claim 1, wherein if the patient has iron stores that are
lower than the normal range for patients of similar age and sex, the
patient is treated with an iron supplement prior to dosing with the
activin-ActRIIa antagonist.

14. The method of claim 1, wherein if the patient has a transferrin
saturation of less than 20%, the patient is treated with an iron
supplement prior to dosing with the activin-ActRIIa antagonist.

15. The method of claim 1, wherein if the patient has a ferritin level of
less than 100 ng/ml, the patient is treated with an iron supplement prior
to dosing with the activin-ActRIIa antagonist.

16. The method of claim 1, wherein monitoring red blood cell levels
comprises monitoring one or more of the following: hemoglobin levels and
hematocrit levels.

18. The method of claim 1, wherein monitoring iron stores comprises
monitoring one or more of the following: transferrin saturation, ferritin
levels or total iron binding capacity.

19. A method for dosing a patient with an activin-ActRIIa antagonist, the
method comprising dosing the patient in amounts and at intervals selected
so as to reduce the risk of causing a rise in hemoglobin levels greater
than 1 g/dl in two weeks.

20. The method of claim 1, wherein the patient is suffering from anemia.

21. The method of claim 1, wherein the patient is in need of bone growth,
increased bone density or increased bone strength.

22. The method of claim 1, wherein the patient is suffering from a
bone-related disorder.

23. The method of claim 1, wherein the patient has cancer.

24. The method of claim 23, wherein the patient has breast cancer.

25. The method of claim 1, wherein the activin-ActRIIa antagonist is an
antibody that binds to a target protein selected from the group
consisting of:an activin and ActRIIA.

26. The method of claim 1, wherein the activin-ActRIIa antagonist is
inhibin or a conservative variant of inhibin.

27. The method of claim 1, wherein the activin-ActRIIa antagonist is a
protein comprising a follistatin domain that binds to and antagonizes
activin.

28. The method of claim 1, wherein the activin-ActRIIa antagonist is a
protein selected from the group consisting of: follistatin,
follastatin-related gene (FLRG) and a conservative variant of the
forgoing.

30. The method of claim 29, wherein the polypeptide has one or more of the
following characteristics:i) binds to an ActRIIa ligand with a KD of at
least 10.sup.-7 M; andii) inhibits ActRIIa signaling in a cell.

31. The method of claim 29, wherein said polypeptide is a fusion protein
including, in addition to an ActRIIa polypeptide domain, one or more
polypeptide portions that enhance one or more of in vivo stability, in
vivo half life, uptake/administration, tissue localization or
distribution, formation of protein complexes, and/or purification.

32. The method of claim 29, wherein said fusion protein includes a
polypeptide portion selected from the group consisting of: an
immunoglobulin Fc domain and a serum albumin.

33. The method of claim 29, wherein said polypeptide includes one or more
modified amino acid residues selected from: a glycosylated amino acid, a
PEGylated amino acid, a farnesylated amino acid, an acetylated amino
acid, a biotinylated amino acid, an amino acid conjugated to a lipid
moiety, and an amino acid conjugated to an organic derivatizing agent.

35. A method for administering an ActRIIa-Fc fusion protein to a patient,
the method comprising administering the ActRIIa-Fc fusion protein no more
frequently than once per 60 days.

36. The method of claim 35, wherein the activin antagonist is administered
to the patient no more frequently than once per 90 days.

37. The method of claim 35, wherein the activin antagonist is administered
to the patient no more frequently than once per 120 days.

38. A method for administering an ActRIIa-Fc fusion protein to a patient
in need thereof, the method comprising administering the ActRIIa-Fc
fusion protein to a patient having a hemoglobin level of less than 10
g/dL.

39. The method of claim 38, wherein the patient has a hemoglobin level of
less than 11 g/dL.

40. The method of claim 38, wherein the patient has a hemoglobin level of
less than 12 g/dL.

41. The method of claim 38, wherein the patient is in need of bone growth,
increased bone density or increased bone strength.

42. The method of claim 38, wherein the patient is suffering from a
bone-related disorder.

43. The method of claim 38, wherein the patient has cancer.

44. The method of claim 38, wherein the patient has breast cancer.

Description:

RELATED APPLICATIONS

[0001]This application claims the benefit of U.S. Provisional Application
No. 61/133,354, filed on Jun. 26, 2008, the specification of which is
incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002]The transforming growth factor-beta (TGF-beta) superfamily contains
a variety of growth factors that share common sequence elements and
structural motifs. These proteins are known to exert biological effects
on a large variety of cell types in both vertebrates and invertebrates.
Members of the superfamily perform important functions during embryonic
development in pattern formation and tissue specification and can
influence a variety of differentiation processes, including adipogenesis,
myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis,
and epithelial cell differentiation. The family is divided into two
general branches: the BMP/GDF and the TGF-beta/Activin/BMP10 branches,
whose members have diverse, often complementary effects. By manipulating
the activity of a member of the TGF-beta family, it is often possible to
cause significant physiological changes in an organism. For example, the
Piedmontese and Belgian Blue cattle breeds carry a loss-of-function
mutation in the GDF8 (also called myostatin) gene that causes a marked
increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4.
Furthermore, in humans, inactive alleles of GDF8 are associated with
increased muscle mass and, reportedly, exceptional strength. Schuelke et
al., N Engl J Med 2004, 350:2682-8.

[0003]Changes in muscle, bone, cartilage and other tissues may be achieved
by agonizing or antagonizing signaling that is mediated by an appropriate
TGF-beta family member. It is an object of the present disclosure to
provide alternative methods for administering modulators of the TGF-beta
superfamily to patients.

SUMMARY OF THE INVENTION

[0004]In part, the disclosure relates to methods for administering activin
antagonists, as well as ActRIIa antagonists (collectively,
"activin-ActRIIa antagonists"), to patients in a manner that is
appropriate given the effects that such antagonists can have on a variety
of tissues, including red blood cells. In part, the disclosure
demonstrates that activin-ActRIIa antagonists can increase red blood cell
and hemoglobin levels and also increase bone density. This dual effect
has particular advantages in patients that have both anemia and bone
loss, such as many cancer patients (where anemia and bone loss can be a
consequence of the tumor or a consequence of irradiation or
chemotherapy), patients with osteoporosis and patients with renal
failure. In particular, the disclosure demonstrates that a soluble form
of ActRIIa acts as an inhibitor of activin and, when administered in
vivo, increases red blood cell levels. While soluble ActRIIa may affect
red blood cell levels through a mechanism other than activin antagonism,
the disclosure nonetheless demonstrates that desirable therapeutic agents
may be selected on the basis of activin antagonism or ActRIIa antagonism
or both. Such agents are referred to collectively as activin-ActRIIa
antagonists. As described herein, and in published patent applications
WO/2009/038745, WO/2008/100384, WO/2008/094708, WO/2008/076437,
WO/2007/062188 and WO/2006/012627, activin-ActRIIa antagonists also have
a variety of other therapeutic uses including, for example, promoting
bone growth, decreasing FSH levels, treating multiple myeloma and
treating breast cancer. In certain instances, when administering an
activin-ActRIIa antagonists for promoting bone growth or treating breast
cancer, it may be desirable to monitor the effects on red blood cells
during administration of an activin-ActRIIa antagonists, or to determine
or adjust the dosing of an activin-ActRIIa antagonists, in order to
reduce undesired effects on red blood cells. For example, excessive
increases in red blood cell levels, hemoglobin levels, or hematocrit
levels may cause increases in blood pressure or other undesirable side
effects. It may also be desirable to restrict dosing of activin-ActRIIa
antagonists to patients who have appropriate hematologic parameters. For
example, it may be desirable to limit dosing to only those patients who
have a hemoglobin level below normal (e.g., below 12 g/dL, below 11 g/dL,
below 10 g/dL or below 9 g/dL or lower).

[0005]Therefore, in certain embodiments, the disclosure provides methods
for managing a patient that has been treated with, or is a candidate to
be treated with, an activin-ActRIIa antagonist, including, for example,
activin-binding ActRIIa polypeptides, anti-activin antibodies,
anti-ActRIIa antibodies, activin- or ActRIIa-targeted small molecules and
aptamers, and nucleic acids that decrease expression of activin or
ActRIIa, by monitoring in the patient one or more hematologic parameters
that correlate with an increase in red blood cell levels, such as, for
example, red blood cell levels, blood pressure, or iron stores.

[0006]In certain aspects, the disclosure provides polypeptides comprising
a soluble, activin-binding ActRIIa polypeptide that binds to activin.
ActRIIa polypeptides may be formulated as a pharmaceutical preparation
comprising the activin-binding ActRIIa polypeptide and a pharmaceutically
acceptable carrier. The activin-binding ActRIIa polypeptide may bind to
activin with a KD less than 1 micromolar or less than 100, 10 or 1
nanomolar. Optionally, the activin-binding ActRIIa polypeptide
selectively binds activin versus GDF11 and/or GDF8, and optionally with a
KD that is at least 10-fold, 20-fold or 50-fold lower with respect to
activin than with respect to GDF11 and/or GDF8. While not wishing to be
bound to a particular mechanism of action, it is expected that this
degree of selectivity for activin inhibition over GDF11/GDF8 inhibition
in ActRIIa-Fc accounts for effects on bone or erythropoiesis without a
consistently measurable effect on muscle. In many embodiments, an ActRIIa
polypeptide will be selected for causing less than 15%, less than 10% or
less than 5% increase in muscle at doses that achieve desirable effects
on red blood cell levels. In other embodiments, the effect on muscle is
acceptable and need not be selected against. The composition may be at
least 95% pure, with respect to other polypeptide components, as assessed
by size exclusion chromatography, and optionally, the composition is at
least 98% pure. An activin-binding ActRIIa polypeptide for use in such a
preparation may be any of those disclosed herein, such as a polypeptide
having (i.e. comprising) an amino acid sequence selected from SEQ ID NOs:
2, 3, 7, 12 or 13, or having (i.e. comprising) an amino acid sequence
that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino
acid sequence selected from SEQ ID NOs: 2, 3, 7, 12 or 13. An
activin-binding ActRIIa polypeptide may include a functional fragment of
a natural ActRIIa polypeptide, such as one comprising at least 10, 20 or
30 amino acids of a sequence selected from SEQ ID NOs: 1-3 or a sequence
of SEQ ID NO: 2, lacking the C-terminal 10 to 15 amino acids (the
"tail").

[0007]A soluble, activin-binding ActRIIa polypeptide may include one or
more alterations in the amino acid sequence (e.g., in the ligand-binding
domain) relative to a naturally occurring ActRIIa polypeptide. Examples
of altered ActRIIa polypeptides are provided in WO 2006/012627, pp. 59-60
and pp. 55-58, respectively, which is incorporated by reference herein,
and throughout U.S. patent application Ser. No. 12/012,652, incorporated
by reference herein. The alteration in the amino acid sequence may, for
example, alter glycosylation of the polypeptide when produced in a
mammalian, insect or other eukaryotic cell or alter proteolytic cleavage
of the polypeptide relative to the naturally occurring ActRIIa
polypeptide.

[0008]An activin-binding ActRIIa polypeptide may be a fusion protein that
has, as one domain, an ActRIIa polypeptide, (e.g., a ligand-binding
portion of an ActRIIa) and one or more additional domains that provide a
desirable property, such as improved pharmacokinetics, easier
purification, targeting to particular tissues, etc. For example, a domain
of a fusion protein may enhance one or more of in vivo stability, in vivo
half life, uptake/administration, tissue localization or distribution,
formation of protein complexes, multimerization of the fusion protein,
and/or purification. An activin-binding ActRIIa fusion protein may
include an immunoglobulin Fc domain (wild-type or mutant) or a serum
albumin or other polypeptide portion that provides desirable properties
such as improved pharmacokinetics, improved solubility or improved
stability. In a preferred embodiment, an ActRIIa-Fc fusion comprises a
relatively unstructured linker positioned between the Fc domain and the
extracellular ActRIIa domain. This unstructured linker may be an
artificial sequence of 1, 2, 3, 4 or 5 amino acids or a length of between
5 and 15, 20, 30, 50 or more amino acids that are relatively free of
secondary structure, or a mixture of both. A linker may be rich in
glycine and proline residues and may, for example, contain a single
sequence of threonine/serine and glycines or repeating sequences of
threonine/serine and glycines (e.g., TG4 (SEQ ID NO: 15) or SG4
(SEQ ID NO: 16) singlets or repeats). A fusion protein may include a
purification subsequence, such as an epitope tag, a FLAG tag, a
polyhistidine sequence, and a GST fusion. Optionally, a soluble ActRIIa
polypeptide includes one or more modified amino acid residues selected
from: a glycosylated amino acid, a PEGylated amino acid, a famesylated
amino acid, an acetylated amino acid, a biotinylated amino acid, an amino
acid conjugated to a lipid moiety, and an amino acid conjugated to an
organic derivatizing agent. A pharmaceutical preparation may also include
one or more additional compounds such as a compound that is used to treat
a bone disorder or a compound that is used to treat anemia. Preferably, a
pharmaceutical preparation is substantially pyrogen free. In general, it
is preferable that an ActRIIa protein be expressed in a mammalian cell
line that mediates suitably natural glycosylation of the ActRIIa protein
so as to diminish the likelihood of an unfavorable immune response in a
patient. Human and CHO cell lines have been used successfully, and it is
expected that other common mammalian expression systems will be useful.

[0009]As described herein, ActRIIa proteins designated ActRIIa-Fc have
desirable properties, including selective binding to activin versus GDF8
and/or GDF11, high affinity ligand binding and serum half life greater
than two weeks in animal models and in human patients. In certain
embodiments the invention provides ActRIIa-Fc polypeptides and
pharmaceutical preparations comprising such polypeptides and a
pharmaceutically acceptable excipient.

[0010]In certain aspects, the disclosure provides nucleic acids encoding a
soluble activin-binding ActRIIa polypeptide. An isolated polynucleotide
may comprise a coding sequence for a soluble, activin-binding ActRIIa
polypeptide, such as described above. For example, an isolated nucleic
acid may include a sequence coding for an extracellular domain (e.g.,
ligand-binding domain) of an ActRIIa and a sequence that would code for
part or all of the transmembrane domain and/or the cytoplasmic domain of
an ActRIIa, but for a stop codon positioned within the transmembrane
domain or the cytoplasmic domain, or positioned between the extracellular
domain and the transmembrane domain or cytoplasmic domain. For example,
an isolated polynucleotide may comprise a full-length ActRIIa
polynucleotide sequence such as SEQ ID NO: 4 or a partially truncated
version of ActRIIa, such as a nucleic acid comprising the nucleic acid
sequence of SEQ ID NO:5, which corresponds to the extracellular domain of
ActRIIa. An isolated polynucleotide may further comprise a transcription
termination codon at least six hundred nucleotides before the 3'-terminus
or otherwise positioned such that translation of the polynucleotide gives
rise to an extracellular domain optionally fused to a truncated portion
of a full-length ActRIIa. A preferred nucleic acid sequence for ActRIIa
is SEQ ID NO:14. Nucleic acids disclosed herein may be operably linked to
a promoter for expression, and the disclosure provides cells transformed
with such recombinant polynucleotides. Preferably the cell is a mammalian
cell such as a CHO cell.

[0011]In certain aspects, the disclosure provides methods for making a
soluble, activin-binding ActRIIa polypeptide. Such a method may include
expressing any of the nucleic acids (e.g., SEQ ID NO: 4, 5 or 14)
disclosed herein in a suitable cell, such as a Chinese hamster ovary
(CHO) cell or human cell. Such a method may comprise: a) culturing a cell
under conditions suitable for expression of the soluble ActRIIa
polypeptide, wherein said cell is transformed with a soluble ActRIIa
expression construct; and b) recovering the soluble ActRIIa polypeptide
so expressed. Soluble ActRIIa polypeptides may be recovered as crude,
partially purified or highly purified fractions. Purification may be
achieved by a series of purification steps, including, for example, one,
two or three or more of the following, in any order: protein A
chromatography, anion exchange chromatography (e.g., Q sepharose),
hydrophobic interaction chromatography (e.g., phenylsepharose), size
exclusion chromatography, and cation exchange chromatography. Soluble
ActRIIa polypeptides may be formulated in liquid or solid (e.g.,
lyophilized) forms.

[0012]In certain aspects, the disclosure provides a method for dosing a
patient with an activin-ActRIIa antagonist, comprising dosing the patient
in amounts and at intervals selected so as to reduce the risk of causing
a rise in hemoglobin levels greater than 0.5 g/dL, 1 g/dl or 1.5 g/dL in
two weeks.

[0013]In certain aspects, the disclosure provides a method for
administering an ActRIIa-Fc fusion protein to a patient, comprising
administering the ActRIIa fusion protein no more frequently than once per
60 days, once per 90 days, or once per 120 days. In certain embodiments,
the patient may be a patient in need of bone growth or a patient
suffering from or at risk for developing breast cancer or multiple
myeloma, or a patient in need of having decreased FSH.

[0015]FIG. 2 shows the binding of ActRIIa-hFc to activin and GDF-11, as
measured by BiaCore® assay.

[0016]FIG. 3 shows the effects of ActRIIa-hFc on red blood cell counts in
female non-human primates. Female cynomolgus monkeys (four groups of five
monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg
of ActRIIa-hFc on day 0, day 7, day 14 and day 21. FIG. 3A shows red
blood cell (RBC) counts. FIG. 3B shows hemoglobin levels. Statistical
significance is relative to baseline for each treatment group. At day 57,
two monkeys remained in each group.

[0017]FIG. 4 shows the effects of ActRIIa-hFc on red blood cell counts in
male non-human primates. Male cynomolgus monkeys (four groups of five
monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg
of ActRIIa-hFc on day 0, day 7, day 14 and day 21. FIG. 4A shows red
blood cell (RBC) counts. FIG. 4B shows hemoglobin levels. Statistical
significance is relative to baseline for each treatment group. At day 57,
two monkeys remained in each group.

[0018]FIG. 5 shows the effects of ActRIIa-hFc on reticulocyte counts in
female non-human primates. Cynomolgus monkeys (four groups of five
monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg
of ActRIIa-hFc on day 0, day 7, day 14 and day 21. FIG. 5A shows absolute
reticulocyte counts. FIG. 5B shows the percentage of reticulocytes
relative to RBCs. Statistical significance is relative to baseline for
each group. At day 57, two monkeys remained in each group.

[0019]FIG. 6 shows the effects of ActRIIa-hFc on reticulocyte counts in
female non-human primates. Cynomolgus monkeys (four groups of five
monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg
of ActRIIa-hFc on day 0, day 7, day 14 and day 21. FIG. 6A shows absolute
reticulocyte counts. FIG. 6B shows the percentage of reticulocytes
relative to RBCs. Statistical significance is relative to baseline for
each group. At day 57, two monkeys remained in each group.

[0020]FIG. 7 shows results from the human clinical trial described in
Example 5, where the area-under-curve (AUC) and administered dose of
ActRIIa-hFc have a linear correlation, regardless of whether ActRIIa-hFc
was administered intravenously (IV) or subcutaneously (SC).

[0021]FIG. 8 shows a comparison of serum levels of ActRIIa-hFc in patients
administered IV or SC.

[0023]FIG. 10 depicts the median change from baseline of hematocrit levels
from the human clinical trial described in Example 5. ActRIIa-hFc was
administered intravenously (IV) at the indicated dosage.

[0024]FIG. 11 depicts the median change from baseline of hemoglobin levels
from the human clinical trial described in Example 5. ActRIIa-hFc was
administered intravenously (IV) at the indicated dosage.

[0025]FIG. 12 depicts the median change from baseline of RBC (red blood
cell) count from the human clinical trial described in Example 5.
ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.

[0026]FIG. 13 depicts the median change from baseline of reticulocyte
count from the human clinical trial described in Example 5. ActRIIa-hFc
was administered intravenously (IV) at the indicated dosage.

[0027]FIG. 14 shows an alignment of human ActRIIA and ActRIIB with the
residues that are deduced herein, based on composite analysis of multiple
ActRIIB and ActRIIA crystal structures to directly contact ligand (the
ligand binding pocket) indicated with boxes.

[0028]FIG. 15 shows the effect of ActRIIA-mFc on hematocrit in a mouse
model of chemotherapy-induced anemia. Data are means±SEM. *, P<0.05
vs. vehicle at same time point. A single dose of ActRIIA-mFc before
chemotherapy prevented the decline in hematocrit level otherwise observed
after administration of the chemotherapeutic paclitaxel.

[0029]FIG. 16 shows the dose-dependent effect of ActRIIA-mFc on hematocrit
in a mouse model of chemotherapy-induced anemia. Data are means±SEM.
**, P<0.01; ***, P<0.001 vs. vehicle at same time point. Two weeks
after paclitaxel administration, ActRIIA-mFc treatment increased
hematocrit level as a function of dose number.

[0030]FIG. 17 shows the effect of ActRIIA-mFc on hematocrit in a partially
nephrectomized (NEPHX) mouse model of chronic kidney disease. Data are
means±SEM. *, P<0.05 vs. vehicle at same time point. ActRIIA-mFc
treatment prevented the decline in hematocrit level otherwise observed at
4 weeks and produced a beneficial trend in hematocrit at 8 weeks.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

[0031]The transforming growth factor-beta (TGF-beta) superfamily contains
a variety of growth factors that share common sequence elements and
structural motifs. These proteins are known to exert biological effects
on a large variety of cell types in both vertebrates and invertebrates.
Members of the superfamily perform important functions during embryonic
development in pattern formation and tissue specification and can
influence a variety of differentiation processes, including adipogenesis,
myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis,
and epithelial cell differentiation. The family is divided into two
general branches: the BMP/GDF and the TGF-beta/Activin/BMP10 branches,
whose members have diverse, often complementary effects. By manipulating
the activity of a member of the TGF-beta family, it is often possible to
cause significant physiological changes in an organism. For example, the
Piedmontese and Belgian Blue cattle breeds carry a loss-of-function
mutation in the GDF8 (also called myostatin) gene that causes a marked
increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4.
Furthermore, in humans, inactive alleles of GDF8 are associated with
increased muscle mass and, reportedly, exceptional strength. Schuelke et
al., N Engl J Med 2004, 350:2682-8.

[0032]Activins are dimeric polypeptide growth factors that belong to the
TGF-beta superfamily. There are three principal activin forms (A, B, and
AB) that are homo/heterodimers of two closely related β subunits
(βAβA, βBβB, and
βAβB, respectively). The human genome also encodes an
activin C and an activin E, which are primarily expressed in the liver,
and heterodimeric forms containing βC or βE are also
known. In the TGF-beta superfamily, activins are unique and
multifunctional factors that can stimulate hormone production in ovarian
and placental cells, support neuronal cell survival, influence cell-cycle
progress positively or negatively depending on cell type, and induce
mesodermal differentiation at least in amphibian embryos (DePaolo et al.,
1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997, Curr Biol.
7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). Moreover,
erythroid differentiation factor (EDF) isolated from the stimulated human
monocytic leukemic cells was found to be identical to activin A (Murata
et al., 1988, PNAS, 85:2434). It has been suggested that activin A
promotes erythropoiesis in the bone marrow. In several tissues, activin
signaling is antagonized by its related heterodimer, inhibin. For
example, during the release of follicle-stimulating hormone (FSH) from
the pituitary, activin promotes FSH secretion and synthesis, while
inhibin prevents FSH secretion and synthesis. Other proteins that may
regulate activin bioactivity and/or bind to activin include follistatin
(FS), follistatin-related protein (FSRP) and α2-macroglobulin.

[0033]TGF-β signals are mediated by heteromeric complexes of type I
and type II serine/threonine kinase receptors, which phosphorylate and
activate downstream Smad proteins upon ligand stimulation (Massague,
2000, Nat. Rev. Mol. Cell Biol. 1:169-178). These type I and type II
receptors are transmembrane proteins, composed of a ligand-binding
extracellular domain with cysteine-rich region, a transmembrane domain,
and a cytoplasmic domain with predicted serine/threonine specificity.
Type I receptors are essential for signaling; and type II receptors are
required for binding ligands and for expression of type I receptors. Type
I and II activin receptors form a stable complex after ligand binding,
resulting in phosphorylation of type I receptors by type II receptors.

[0035]As demonstrated herein, a soluble ActRIIa polypeptide (sActRIIa),
which shows substantial preference in binding to activin A as opposed to
other TGF-beta family members, such as GDF8 or GDF11, is effective to
increase red blood cell levels in vivo. While not wishing to be bound to
any particular mechanism, it is expected that the effect of sActRIIa is
caused primarily by an activin antagonist effect, given the very strong
activin binding (picomolar dissociation constant) exhibited by the
particular sActRIIa construct used in these studies. Regardless of
mechanism, it is apparent from this disclosure that ActRIIa-activin
antagonists increase red blood cell levels in rodents, monkeys and
humans. It should be noted that hematopoiesis is a complex process,
regulated by a variety of factors, including erythropoietin, G-CSF and
iron homeostasis. The terms "increase red blood cell levels" and "promote
red blood cell formation" refer to clinically observable metrics, such as
hematocrit, red blood cell counts and hemoglobin measurements, and are
intended to be neutral as to the mechanism by which such changes occur.

[0036]The data reported herein with respect to non-human primates are
reproducible in mice, rats and humans as well, and therefore, this
disclosure provides methods for using ActRIIa polypeptides and other
activin-ActRIIa antagonists to promote red blood cell production and
increase red blood cell levels in mammals ranging from rodents to humans.

[0037]In addition to stimulating red blood cell levels, activin-ActRIIa
antagonists are useful for a variety of therapeutic applications,
including, for example, promoting bone growth (see PCT Publication No.
WO2007/062188, which is hereby incorporated by reference in its
entirety), and treating breast cancer (see PCT Application No.
PCT/US2008/001429, which is hereby incorporated by reference in its
entirety). In certain instances, when administering an activin-ActRIIa
antagonists for the purpose of increasing bone or treating breast cancer,
it may be desirable to reduce or minimize or otherwise monitor effects on
red blood cells. In some instances, a dual effect on blood cells and bone
or other tissue will be desirable, but it is generally recognized that
pharmaceutically promoted increases in red blood cells, even up to a
level that is typically considered normal, can have adverse effects on
patients, and thus are often monitored or managed with care. By
monitoring various hematologic parameters in patients being treated with,
or who are candidates for treatment with, an activin-ActRIIa antagonist,
appropriate dosing (including amounts and frequency of administration)
may be determined based on an individual patient's needs, baseline
hematologic parameters, and purpose for treatment. Furthermore,
therapeutic progress and effects on one or more hematologic parameters
over time may be useful in managing patients being dosed with an
activin-ActRIIa antagonist by facilitating patient care, determining
appropriate maintenance dosing (both amounts and frequency), etc.

[0038]Activin-ActRIIa antagonists include, for example, activin-binding
soluble ActRIIa polypeptides, antibodies that bind to activin
(particularly the activin A or B subunits, also referred to as
βA or βB) and disrupt ActRIIa binding, antibodies
that bind to ActRIIa and disrupt activin binding, non-antibody proteins
selected for activin or ActRIIa binding (see e.g., WO/2002/088171,
WO/2006/055689, and WO/2002/032925 for examples of such proteins and
methods for design and selection of same), randomized peptides selected
for activin or ActRIIa binding, often affixed to an Fc domain. Two
different proteins (or other moieties) with activin or ActRIIa binding
activity, especially activin binders that block the type I (e.g., a
soluble type I activin receptor) and type II (e.g., a soluble type II
activin receptor) binding sites, respectively, may be linked together to
create a bifunctional binding molecule. Nucleic acid aptamers, small
molecules and other agents that inhibit the activin-ActRIIa signaling
axis are included as activin-ActRIIa antagonists. Various proteins have
activin-ActRIIa antagonist activity, including inhibin (i.e., inhibin
alpha subunit), although inhibin does not universally antagonize activin
in all tissues, follistatin (e.g., follistatin-288 and follistatin-315),
FSRP, FLRG, activin C, alpha(2)-macroglobulin, and an M108A (methionine
to alanine change at position 108) mutant activin A. Generally,
alternative forms of activin, particularly those with alterations in the
type I receptor binding domain can bind to type II receptors and fail to
form an active ternary complex, thus acting as antagonists. Additionally,
nucleic acids, such as antisense molecules, siRNAs or ribozymes that
inhibit activin A, B, C or E, or, particularly, ActRIIa expression, can
be used as activin-ActRIIa antagonists. The activin-ActRIIa antagonist to
be used may exhibit selectivity for inhibiting activin-mediated signaling
versus other members of the TGF-beta family, and particularly with
respect to GDF8 and GDF11.

[0039]The terms used in this specification generally have their ordinary
meanings in the art, within the context of this invention and in the
specific context where each term is used. Certain terms are discussed
below or elsewhere in the specification, to provide additional guidance
to the practitioner in describing the compositions and methods of the
invention and how to make and use them. The scope or meaning of any use
of a term will be apparent from the specific context in which the term is
used.

[0040]"About" and "approximately" shall generally mean an acceptable
degree of error for the quantity measured given the nature or precision
of the measurements. Typically, exemplary degrees of error are within 20
percent (%), preferably within 10%, and more preferably within 5% of a
given value or range of values.

[0041]Alternatively, and particularly in biological systems, the terms
"about" and "approximately" may mean values that are within an order of
magnitude, preferably within 5-fold and more preferably within 2-fold of
a given value. Numerical quantities given herein are approximate unless
stated otherwise, meaning that the term "about" or "approximately" can be
inferred when not expressly stated.

[0042]The methods of the invention may include steps of comparing
sequences to each other, including wild-type sequence to one or more
mutants (sequence variants). Such comparisons typically comprise
alignments of polymer sequences, e.g., using sequence alignment programs
and/or algorithms that are well known in the art (for example, BLAST,
FASTA and MEGALIGN, to name a few). The skilled artisan can readily
appreciate that, in such alignments, where a mutation contains a residue
insertion or deletion, the sequence alignment will introduce a "gap"
(typically represented by a dash, or "A") in the polymer sequence not
containing the inserted or deleted residue.

[0043]"Homologous," in all its grammatical forms and spelling variations,
refers to the relationship between two proteins that possess a "common
evolutionary origin," including proteins from superfamilies in the same
species of organism, as well as homologous proteins from different
species of organism. Such proteins (and their encoding nucleic acids)
have sequence homology, as reflected by their sequence similarity,
whether in terms of percent identity or by the presence of specific
residues or motifs and conserved positions.

[0044]The term "sequence similarity," in all its grammatical forms, refers
to the degree of identity or correspondence between nucleic acid or amino
acid sequences that may or may not share a common evolutionary origin.

[0045]However, in common usage and in the instant application, the term
"homologous," when modified with an adverb such as "highly," may refer to
sequence similarity and may or may not relate to a common evolutionary
origin.

2. ActRIIa Polypeptides

[0046]In certain aspects, the present invention relates to ActRIIa
polypeptides. As used herein, the term "ActRIIa" refers to a family of
activin receptor type IIa (ActRIIa) proteins from any species and
variants derived from such ActRIIa proteins by mutagenesis or other
modification. Reference to ActRIIa herein is understood to be a reference
to any one of the currently identified forms. Members of the ActRIIa
family are generally transmembrane proteins, composed of a ligand-binding
extracellular domain with a cysteine-rich region, a transmembrane domain,
and a cytoplasmic domain with predicted serine/threonine kinase activity.

[0047]The term "ActRIIa polypeptide" includes polypeptides comprising any
naturally occurring polypeptide of an ActRIIa family member as well as
any variants thereof (including mutants, fragments, fusions, and
peptidomimetic forms) that retain a useful activity. See, for example,
WO/2006/012627. For example, ActRIIa polypeptides include polypeptides
derived from the sequence of any known ActRIIa having a sequence at least
about 80% identical to the sequence of an ActRIIa polypeptide, and
optionally at least 85%, 90%, 95%, 97%, 99% or greater identity. For
example, an ActRIIapolypeptide of the invention may bind to and inhibit
the function of an ActRIIa protein and/or activin. An ActRIIa polypeptide
may be selected for activity in promoting red blood cell formation in
vivo. Examples of ActRIIa polypeptides include human ActRIIa precursor
polypeptide (SEQ ID NO: 1) and soluble human ActRIIa polypeptides (e.g.,
SEQ ID NOs: 2, 3, 7 and 12).

[0054]In a specific embodiment, the invention relates to soluble ActRIIa
polypeptides. As described herein, the term "soluble ActRIIa polypeptide"
generally refers to polypeptides comprising an extracellular domain of an
ActRIIa protein. The term "soluble ActRIIa polypeptide," as used herein,
includes any naturally occurring extracellular domain of an ActRIIa
protein as well as any variants thereof (including mutants, fragments and
peptidomimetic forms). An activin-binding ActRIIa polypeptide is one that
retains the ability to bind to activin, including, for example, activin
AA, AB, BB, or forms that include a C or E subunit. Optionally, an
activin-binding ActRIIa polypeptide will bind to activin AA with a
dissociation constant of 1 nM or less. The extracellular domain of an
ActRIIa protein binds to activin and is generally soluble in
physiological conditions, and thus can be termed a soluble,
activin-binding ActRIIa polypeptide. Examples of soluble, activin-binding
ActRIIa polypeptides include the soluble polypeptides illustrated in SEQ
ID NOs: 2, 3, 7, 12 and 13. SEQ ID NO:7 is referred to as ActRIIa-hFc,
and is described further in the Examples. Other examples of soluble,
activin-binding ActRIIa polypeptides comprise a signal sequence in
addition to the extracellular domain of an ActRIIa protein, for example,
the honey bee mellitin leader sequence (SEQ ID NO: 8), the tissue
plaminogen activator (TPA) leader (SEQ ID NO: 9) or the native ActRIIa
leader (SEQ ID NO: 10). The ActRIIa-hFc polypeptide illustrated in SEQ ID
NO:13 uses a TPA leader.

[0055]A general formula for an active ActRIIa variant protein is one that
comprises amino acids 12-82 of SEQ ID NO: 2, respectively, but optionally
beginning at a position ranging from 1-5 or 3-5 and ending at a position
ranging from 110-116 or 110-115, respectively, and comprising no more
than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand
binding pocket, and zero, one or more non-conservative alterations at
positions 40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket. Such
a protein may comprise an amino acid sequence that retains greater than
80%, 90%, 95% or 99% sequence identity to the sequence of amino acids
29-109 of SEQ ID NO: 2.

[0056]Functionally active fragments of ActRIIa polypeptides can be
obtained by screening polypeptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding an ActRIIa
polypeptide. In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid phase
f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or
by chemical synthesis) and tested to identify those peptidyl fragments
that can function as antagonists (inhibitors) of ActRIIa protein or
signaling mediated by activin.

[0057]Functionally active variants of ActRIIa polypeptides can be obtained
by screening libraries of modified polypeptides recombinantly produced
from the corresponding mutagenized nucleic acids encoding an ActRIIa
polypeptide. The variants can be produced and tested to identify those
that can function as antagonists (inhibitors) of ActRIIa protein or
signaling mediated by activin. In certain embodiments, a functional
variant of the ActRIIa polypeptides comprises an amino acid sequence that
is at least 75% identical to an amino acid sequence selected from SEQ ID
NOs: 2 or 3. In certain cases, the functional variant has an amino acid
sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to
an amino acid sequence selected from SEQ ID NOs: 2 or 3.

[0058]Functional variants may be generated by modifying the structure of
an ActRIIa polypeptide for such purposes as enhancing therapeutic
efficacy, or stability (e.g., ex vivo shelf life and resistance to
proteolytic degradation in vivo). Such modified ActRIIa polypeptides when
selected to retain activin binding, are considered functional equivalents
of the naturally-occurring ActRIIa polypeptides. Modified ActRIIa
polypeptides can also be produced, for instance, by amino acid
substitution, deletion, or addition. For instance, it is reasonable to
expect that an isolated replacement of a leucine with an isoleucine or
valine, an aspartate with a glutamate, a threonine with a serine, or a
similar replacement of an amino acid with a structurally related amino
acid (e.g., conservative mutations) will not have a major effect on the
biological activity of the resulting molecule. Conservative replacements
are those that take place within a family of amino acids that are related
in their side chains. Whether a change in the amino acid sequence of an
ActRIIa polypeptide results in a functional homolog can be readily
determined by assessing the ability of the variant ActRIIa polypeptide to
produce a response in cells in a fashion similar to the wild-type ActRIIa
polypeptide.

[0059]In certain embodiments, the present invention contemplates specific
mutations of the ActRIIa polypeptides so as to alter the glycosylation of
the polypeptide. Such mutations may be selected so as to introduce or
eliminate one or more glycosylation sites, such as O-linked or N-linked
glycosylation sites. Asparagine-linked glycosylation recognition sites
generally comprise a tripeptide sequence, asparagine-X-threonine or
asparagine-X-serine (where "X" is any amino acid) which is specifically
recognized by appropriate cellular glycosylation enzymes. The alteration
may also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the wild-type ActRIIa
polypeptide (for O-linked glycosylation sites). A variety of amino acid
substitutions or deletions at one or both of the first or third amino
acid positions of a glycosylation recognition site (and/or amino acid
deletion at the second position) results in non-glycosylation at the
modified tripeptide sequence. Another means of increasing the number of
carbohydrate moieties on an ActRIIa polypeptide is by chemical or
enzymatic coupling of glycosides to the ActRIIa polypeptide. Depending on
the coupling mode used, the sugar(s) may be attached to (a) arginine and
histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as
those of cysteine; (d) free hydroxyl groups such as those of serine,
threonine, or hydroxyproline; (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan; or (f) the amide group of
glutamine. Removal of one or more carbohydrate moieties present on an
ActRIIa polypeptide may be accomplished chemically and/or enzymatically.
Chemical deglycosylation may involve, for example, exposure of the
ActRIIa polypeptide to the compound trifluoromethanesulfonic acid, or an
equivalent compound. This treatment results in the cleavage of most or
all sugars except the linking sugar (N-acetylglucosamine or
N-acetylgalactosamine), while leaving the amino acid sequence intact.
Enzymatic cleavage of carbohydrate moieties on ActRIIa polypeptides can
be achieved by the use of a variety of endo- and exo-glycosidases as
described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence
of an ActRIIa polypeptide may be adjusted, as appropriate, depending on
the type of expression system used, as mammalian, yeast, insect and plant
cells may all introduce differing glycosylation patterns that can be
affected by the amino acid sequence of the peptide. In general, ActRIIa
proteins for use in humans may be expressed in a mammalian cell line that
provides proper glycosylation, such as HEK293 or CHO cell lines, although
other mammalian expression cell lines are expected to be useful as well.
Other non-mammalian cell lines may be used (e.g., yeast, E. coli, insect
cells), and in some cases, such cell lines may be engineered to include
enzymes that confer mammalian-type glycosylation patterns on the
expressed proteins.

[0060]This disclosure further contemplates a method of generating mutants,
particularly sets of combinatorial mutants of an ActRIIa polypeptide, as
well as truncation mutants; pools of combinatorial mutants are especially
useful for identifying functional variant sequences. The purpose of
screening such combinatorial libraries may be to generate, for example,
ActRIIa polypeptide variants which bind to activin or other ligands. A
variety of screening assays are provided below, and such assays may be
used to evaluate variants. For example, an ActRIIa polypeptide variant
may be screened for ability to bind to an ActRIIa ligand, to prevent
binding of an ActRIIa ligand to an ActRIIa polypeptide or to interfere
with signaling caused by an ActRIIa ligand.

[0061]The activity of an ActRIIa polypeptide or its variants may also be
tested in a cell-based or in vivo assay. For example, the effect of an
ActRIIa polypeptide variant on the expression of genes involved in
hematopoiesis may be assessed. This may, as needed, be performed in the
presence of one or more recombinant ActRIIa ligand proteins (e.g.,
activin), and cells may be transfected so as to produce an ActRIIa
polypeptide and/or variants thereof, and optionally, an ActRIIa ligand.
Likewise, an ActRIIa polypeptide may be administered to a mouse or other
animal, and one or more blood measurements, such as an RBC count,
hemoglobin, or reticulocyte count may be assessed.

[0062]Combinatorially-derived variants can be generated which have a
selective or generally increased potency relative to a naturally
occurring ActRIIa polypeptide. Likewise, mutagenesis can give rise to
variants which have intracellular half-lives dramatically different than
the corresponding a wild-type ActRIIa polypeptide. For example, the
altered protein can be rendered either more stable or less stable to
proteolytic degradation or other cellular processes which result in
destruction of, or otherwise inactivation of a native ActRIIa
polypeptide. Such variants, and the genes which encode them, can be
utilized to alter ActRIIa polypeptide levels by modulating the half-life
of the ActRIIa polypeptides. For instance, a short half-life can give
rise to more transient biological effects and, when part of an inducible
expression system, can allow tighter control of recombinant ActRIIa
polypeptide levels within the cell. In an Fc fusion protein, mutations
may be made in the linker (if any) and/or the Fc portion to alter the
half-life of the protein.

[0063]A combinatorial library may be produced by way of a degenerate
library of genes encoding a library of polypeptides which each include at
least a portion of potential ActRIIa polypeptide sequences. For instance,
a mixture of synthetic oligonucleotides can be enzymatically ligated into
gene sequences such that the degenerate set of potential ActRIIa
polypeptide nucleotide sequences are expressible as individual
polypeptides, or alternatively, as a set of larger fusion proteins (e.g.,
for phage display).

[0066]A wide range of techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations and
truncations, and, for that matter, for screening cDNA libraries for gene
products having a certain property. Such techniques will be generally
adaptable for rapid screening of the gene libraries generated by the
combinatorial mutagenesis of ActRIIa polypeptides. The most widely used
techniques for screening large gene libraries typically comprises cloning
the gene library into replicable expression vectors, transforming
appropriate cells with the resulting library of vectors, and expressing
the combinatorial genes under conditions in which detection of a desired
activity facilitates relatively easy isolation of the vector encoding the
gene whose product was detected. Preferred assays include activin binding
assays and activin-mediated cell signaling assays.

[0067]In certain embodiments, the ActRIIa polypeptides of the invention
may further comprise post-translational modifications in addition to any
that are naturally present in the ActRIIa polypeptides. Such
modifications include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
As a result, the modified ActRIIa polypeptides may contain non-amino acid
elements, such as polyethylene glycols, lipids, poly- or mono-saccharide,
and phosphates. Effects of such non-amino acid elements on the
functionality of an ActRIIa polypeptide may be tested as described herein
for other ActRIIa polypeptide variants. When an ActRIIa polypeptide is
produced in cells by cleaving a nascent form of the ActRIIa polypeptide,
post-translational processing may also be important for correct folding
and/or function of the protein. Different cells (such as CHO, HeLa, MDCK,
293, W138, NIH-3T3 or HEK293) have specific cellular machinery and
characteristic mechanisms for such post-translational activities and may
be chosen to ensure the correct modification and processing of the
ActRIIa polypeptides.

[0068]In certain aspects, functional variants or modified forms of the
ActRIIa polypeptides include fusion proteins having at least a portion of
the ActRIIa polypeptides and one or more fusion domains. Well known
examples of such fusion domains include, but are not limited to,
polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin,
protein A, protein G, an immunoglobulin heavy chain constant region (Fc),
maltose binding protein (MBP), or human serum albumin. A fusion domain
may be selected so as to confer a desired property. For example, some
fusion domains are particularly useful for isolation of the fusion
proteins by affinity chromatography. For the purpose of affinity
purification, relevant matrices for affinity chromatography, such as
glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used.
Many of such matrices are available in "kit" form, such as the Pharmacia
GST purification system and the QIAexpress system (Qiagen) useful with
(HIS6) fusion partners. As another example, a fusion domain may be
selected so as to facilitate detection of the ActRIIa polypeptides.
Examples of such detection domains include the various fluorescent
proteins (e.g., GFP) as well as "epitope tags," which are usually short
peptide sequences for which a specific antibody is available. Well known
epitope tags for which specific monoclonal antibodies are readily
available include FLAG, influenza virus haemagglutinin (HA), and c-myc
tags. In some cases, the fusion domains have a protease cleavage site,
such as for Factor Xa or Thrombin, which allows the relevant protease to
partially digest the fusion proteins and thereby liberate the recombinant
proteins therefrom. The liberated proteins can then be isolated from the
fusion domain by subsequent chromatographic separation. In certain
preferred embodiments, an ActRIIa polypeptide is fused with a domain that
stabilizes the ActRIIa polypeptide in vivo (a "stabilizer" domain). By
"stabilizing" is meant anything that increases serum half life,
regardless of whether this is because of decreased destruction, decreased
clearance by the kidney, or other pharmacokinetic effect. Fusions with
the Fc portion of an immunoglobulin are known to confer desirable
pharmacokinetic properties on a wide range of proteins. Constant domains
from an immunoglobulin, particularly an IgG heavy chain, may also be used
as stabilizing domains. Likewise, fusions to human serum albumin can
confer desirable properties. Other types of fusion domains that may be
selected include multimerizing (e.g., dimerizing, tetramerizing) domains
and functional domains (that confer an additional biological function,
such as further stimulation of bone growth).

[0070]Optionally, the Fc domain has one or more mutations at residues such
as Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc
domain having one or more of these mutations (e.g., Asp-265 mutation) has
reduced ability of binding to the Fcγ receptor relative to a
wildtype Fc domain. In other cases, the mutant Fc domain having one or
more of these mutations (e.g., Asn-434 mutation) has increased ability of
binding to the MHC class I-related Fc-receptor (FcRN) relative to a
wildtype Fc domain. Fc domains from IgG2, IgG3 and IgG4 may also be used.

[0071]It is understood that different elements of the fusion proteins may
be arranged in any manner that is consistent with the desired
functionality. For example, an ActRIIa polypeptide may be placed
C-terminal to a heterologous domain, or, alternatively, a heterologous
domain may be placed C-terminal to an ActRIIA polypeptide. The ActRIIa
polypeptide domain and the heterologous domain need not be adjacent in a
fusion protein, and additional domains or amino acid sequences may be
included C- or N-terminal to either domain or between the domains.

[0072]In certain embodiments, the ActRIIaa polypeptides of the present
invention contain one or more modifications that are capable of
stabilizing the ActRIIa polypeptides. For example, such modifications
enhance the in vitro half life of the ActRIIa polypeptides, enhance
circulatory half life of the ActRIIa polypeptides or reducing proteolytic
degradation of the ActRIIa polypeptides. Such stabilizing modifications
include, but are not limited to, fusion proteins (including, for example,
fusion proteins comprising an ActRIIa polypeptide and a stabilizer
domain), modifications of a glycosylation site (including, for example,
addition of a glycosylation site to an ActRIIa polypeptide), and
modifications of carbohydrate moiety (including, for example, removal of
carbohydrate moieties from an ActRIIa polypeptide). As used herein, the
term "stabilizer domain" not only refers to a fusion domain (e.g., Fc) as
in the case of fusion proteins, but also includes nonproteinaceous
modifications such as a carbohydrate moiety, or nonproteinaceous moiety,
such as polyethylene glycol.

[0073]In certain embodiments, the present invention makes available
isolated and/or purified forms of the ActRIIa polypeptides, which are
isolated from, or otherwise substantially free of, other proteins.
ActRIIa polypeptides will generally be produced by expression from
recombinant nucleic acids.

3. Nucleic Acids Encoding ActRIIa Polypeptides

[0074]In certain aspects, the invention provides isolated and/or
recombinant nucleic acids encoding any of the ActRIIa polypeptides (e.g.,
soluble ActRIIa polypeptides), including fragments, functional variants
and fusion proteins disclosed herein. For example, SEQ ID NO: 4 encodes
the naturally occurring human ActRIIa precursor polypeptide, while SEQ ID
NO: 5 encodes the processed extracellular domain of ActRIIa. The subject
nucleic acids may be single-stranded or double stranded. Such nucleic
acids may be DNA or RNA molecules. These nucleic acids may be used, for
example, in methods for making ActRIIa polypeptides or as direct
therapeutic agents (e.g., in a gene therapy approach).

[0075]In certain aspects, the subject nucleic acids encoding ActRIIa
polypeptides are further understood to include nucleic acids that are
variants of SEQ ID NO: 4 or 5.

[0076]In certain embodiments, the invention provides isolated or
recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%,
97%, 98%, 99% or 100% identical to SEQ ID NOs: 4 or 5. One of ordinary
skill in the art will appreciate that nucleic acid sequences
complementary to SEQ ID NOs: 4 or 5, and variants of SEQ ID NOs: 4 or 5
are also within the scope of this invention. In further embodiments, the
nucleic acid sequences of the invention can be isolated, recombinant,
and/or fused with a heterologous nucleotide sequence, or in a DNA
library.

[0077]In other embodiments, nucleic acids of the invention also include
nucleotide sequences, and the ActRIIa polypeptides encoded by such
nucleic acids, that hybridize under highly stringent conditions to the
nucleotide sequence designated in SEQ ID NOs: 4 or 5, complement sequence
of SEQ ID NOs: 4 or 5 or fragments thereof. As discussed above, one of
ordinary skill in the art will understand readily that appropriate
stringency conditions which promote DNA hybridization can be varied. One
of ordinary skill in the art will understand readily that appropriate
stringency conditions which promote DNA hybridization can be varied. For
example, one could perform the hybridization at 6.0× sodium
chloride/sodium citrate (SSC) at about 45° C., followed by a wash
of 2.0×SSC at 50° C. For example, the salt concentration in
the wash step can be selected from a low stringency of about
2.0×SSC at 50° C. to a high stringency of about
0.2×SSC at 50° C. In addition, the temperature in the wash
step can be increased from low stringency conditions at room temperature,
about 22° C., to high stringency conditions at about 65° C.
Both temperature and salt may be varied, or temperature or salt
concentration may be held constant while the other variable is changed.
In one embodiment, the invention provides nucleic acids which hybridize
under low stringency conditions of 6×SSC at room temperature
followed by a wash at 2×SSC at room temperature.

[0078]Isolated nucleic acids which differ from the nucleic acids as set
forth in SEQ ID NOs: 4 or 5 due to degeneracy in the genetic code are
also within the scope of the invention. For example, a number of amino
acids are designated by more than one triplet. Codons that specify the
same amino acid, or synonyms (for example, CAU and CAC are synonyms for
histidine) may result in "silent" mutations which do not affect the amino
acid sequence of the protein. However, it is expected that DNA sequence
polymorphisms that do lead to changes in the amino acid sequences of the
subject proteins will exist among mammalian cells. One skilled in the art
will appreciate that these variations in one or more nucleotides (up to
about 3-5% of the nucleotides) of the nucleic acids encoding a particular
protein may exist among individuals of a given species due to natural
allelic variation. Any and all such nucleotide variations and resulting
amino acid polymorphisms are within the scope of this invention.

[0079]In certain embodiments, the recombinant nucleic acids of the
invention may be operably linked to one or more regulatory nucleotide
sequences in an expression construct. Regulatory nucleotide sequences
will generally be appropriate to the host cell used for expression.
Numerous types of appropriate expression vectors and suitable regulatory
sequences are known in the art for a variety of host cells. Typically,
said one or more regulatory nucleotide sequences may include, but are not
limited to, promoter sequences, leader or signal sequences, ribosomal
binding sites, transcriptional start and termination sequences,
translational start and termination sequences, and enhancer or activator
sequences. Constitutive or inducible promoters as known in the art are
contemplated by the invention. The promoters may be either naturally
occurring promoters, or hybrid promoters that combine elements of more
than one promoter. An expression construct may be present in a cell on an
episome, such as a plasmid, or the expression construct may be inserted
in a chromosome. In a preferred embodiment, the expression vector
contains a selectable marker gene to allow the selection of transformed
host cells. Selectable marker genes are well known in the art and will
vary with the host cell used.

[0080]In certain aspects of the invention, the subject nucleic acid is
provided in an expression vector comprising a nucleotide sequence
encoding an ActRIIa polypeptide and operably linked to at least one
regulatory sequence. Regulatory sequences are art-recognized and are
selected to direct expression of the ActRIIa polypeptide. Accordingly,
the term regulatory sequence includes promoters, enhancers, and other
expression control elements. Exemplary regulatory sequences are described
in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic
Press, San Diego, Calif. (1990). For instance, any of a wide variety of
expression control sequences that control the expression of a DNA
sequence when operatively linked to it may be used in these vectors to
express DNA sequences encoding an ActRIIa polypeptide. Such useful
expression control sequences, include, for example, the early and late
promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate
early promoter, RSV promoters, the lac system, the trp system, the TAC or
TRC system, T7 promoter whose expression is directed by T7 RNA
polymerase, the major operator and promoter regions of phage lambda, the
control regions for fd coat protein, the promoter for 3-phosphoglycerate
kinase or other glycolytic enzymes, the promoters of acid phosphatase,
e.g., Pho5, the promoters of the yeast α-mating factors, the
polyhedron promoter of the baculovirus system and other sequences known
to control the expression of genes of prokaryotic or eukaryotic cells or
their viruses, and various combinations thereof. It should be understood
that the design of the expression vector may depend on such factors as
the choice of the host cell to be transformed and/or the type of protein
desired to be expressed. Moreover, the vector's copy number, the ability
to control that copy number and the expression of any other protein
encoded by the vector, such as antibiotic markers, should also be
considered.

[0081]A recombinant nucleic acid of the invention can be produced by
ligating the cloned gene, or a portion thereof, into a vector suitable
for expression in either prokaryotic cells, eukaryotic cells (yeast,
avian, insect or mammalian), or both. Expression vehicles for production
of a recombinant ActRIIa polypeptide include plasmids and other vectors.
For instance, suitable vectors include plasmids of the types:
pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,
pBTac-derived plasmids and pUC-derived plasmids for expression in
prokaryotic cells, such as E. coli.

[0082]Some mammalian expression vectors contain both prokaryotic sequences
to facilitate the propagation of the vector in bacteria, and one or more
eukaryotic transcription units that are expressed in eukaryotic cells.
The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,
pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of
mammalian expression vectors suitable for transfection of eukaryotic
cells. Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug resistance
selection in both prokaryotic and eukaryotic cells. Alternatively,
derivatives of viruses such as the bovine papilloma virus (BPV-1), or
Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for
transient expression of proteins in eukaryotic cells. Examples of other
viral (including retroviral) expression systems can be found below in the
description of gene therapy delivery systems. The various methods
employed in the preparation of the plasmids and in transformation of host
organisms are well known in the art. For other suitable expression
systems for both prokaryotic and eukaryotic cells, as well as general
recombinant procedures, see Molecular Cloning A Laboratory Manual, 3rd
Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press, 2001). In some instances, it may be desirable to express the
recombinant polypeptides by the use of a baculovirus expression system.
Examples of such baculovirus expression systems include pVL-derived
vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors
(such as pAcUWI), and pBlueBac-derived vectors (such as the β-gal
containing pBlueBac III).

[0083]In a preferred embodiment, a vector will be designed for production
of the subject ActRIIa polypeptides in CHO cells, such as a Pcmv-Script
vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,
Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As will
be apparent, the subject gene constructs can be used to cause expression
of the subject ActRIIa polypeptides in cells propagated in culture, e.g.,
to produce proteins, including fusion proteins or variant proteins, for
purification.

[0084]This disclosure also pertains to a host cell transfected with a
recombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 5)
for one or more of the subject ActRIIa polypeptides. The host cell may be
any prokaryotic or eukaryotic cell. For example, an ActRIIa polypeptide
of the invention may be expressed in bacterial cells such as E. coli,
insect cells (e.g., using a baculovirus expression system), yeast, or
mammalian cells. Other suitable host cells are known to those skilled in
the art.

[0085]Accordingly, the present invention further pertains to methods of
producing the subject ActRIIa polypeptides. For example, a host cell
transfected with an expression vector encoding an ActRIIa polypeptide can
be cultured under appropriate conditions to allow expression of the
ActRIIa polypeptide to occur. The ActRIIa polypeptide may be secreted and
isolated from a mixture of cells and medium containing the ActRIIa
polypeptide. Alternatively, the ActRIIa polypeptide may be retained
cytoplasmically or in a membrane fraction and the cells harvested, lysed
and the protein isolated. A cell culture includes host cells, media and
other byproducts. Suitable media for cell culture are well known in the
art. The subject ActRIIa polypeptides can be isolated from cell culture
medium, host cells, or both, using techniques known in the art for
purifying proteins, including ion-exchange chromatography, gel filtration
chromatography, ultrafiltration, electrophoresis, immunoaffinity
purification with antibodies specific for particular epitopes of the
ActRIIa polypeptides and affinity purification with an agent that binds
to a domain fused to the ActRIIa polypeptide (e.g., a protein A column
may be used to purify an ActRIIa-Fc fusion). In a preferred embodiment,
the ActRIIa polypeptide is a fusion protein containing a domain which
facilitates its purification. In a preferred embodiment, purification is
achieved by a series of column chromatography steps, including, for
example, three or more of the following, in any order: protein A
chromatography, Q sepharose chromatography, phenylsepharose
chromatography, size exclusion chromatography, and cation exchange
chromatography. The purification could be completed with viral filtration
and buffer exchange. As demonstrated herein, ActRIIa-hFc protein was
purified to a purity of >98% as determined by size exclusion
chromatography and >95% as determined by SDS PAGE. This level of
purity was sufficient to achieve desirable results in mice, rats,
non-human primates and humans.

[0086]In another embodiment, a fusion gene coding for a purification
leader sequence, such as a poly-(His)/enterokinase cleavage site sequence
at the N-terminus of the desired portion of the recombinant ActRIIa
polypeptide, can allow purification of the expressed fusion protein by
affinity chromatography using a Ni2+ metal resin. The purification
leader sequence can then be subsequently removed by treatment with
enterokinase to provide the purified ActRIIa polypeptide (e.g., see
Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,
PNAS USA 88:8972).

[0087]Techniques for making fusion genes are well known. Essentially, the
joining of various DNA fragments coding for different polypeptide
sequences is performed in accordance with conventional techniques,
employing blunt-ended or stagger-ended termini for ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of
cohesive ends as appropriate, alkaline phosphatase treatment to avoid
undesirable joining, and enzymatic ligation. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of gene
fragments can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments which can
subsequently be annealed to generate a chimeric gene sequence (see, for
example, Current Protocols in Molecular Biology, eds. Ausubel et al.,
John Wiley & Sons: 1992).

4. Alternative Activin and ActRIIa Antagonists

[0088]The data presented herein demonstrates that antagonists of
activin-ActRIIa signaling can be used to increase red blood cell or
hemoglobin levels. Although soluble ActRIIa polypeptides, and
particularly ActRIIa-Fc, are preferred antagonists, and although such
antagonists may affect red blood cell levels through a mechanism other
than activin antagonism (e.g., activin inhibition may be an indicator of
the tendency of an agent to inhibit the activities of a spectrum of
molecules, including, perhaps, other members of the TGF-beta superfamily,
and such collective inhibition may lead to the desired effect on
hematopoiesis), other types of activin-ActRIIa antagonists are expected
to be useful, including anti-activin (e.g., activin βA,
βB, βC and βE) antibodies, anti-ActRIIa
antibodies, antisense, RNAi or ribozyme nucleic acids that inhibit the
production of ActRIIa, and other inhibitors of activin or ActRIIa,
particularly those that disrupt activin-ActRIIa binding.

[0089]An antibody that is specifically reactive with an ActRIIa
polypeptide (e.g., a soluble ActRIIa polypeptide) and which either binds
competitively to ligand with the ActRIIa polypeptide or otherwise
inhibits ActRIIa-mediated signaling may be used as an antagonist of
ActRIIa polypeptide activities. Likewise, an antibody that is
specifically reactive with an activin βA, βB,
βC or βE polypeptide, or any heterodimer thereof, and
which disrupts ActRIIa binding may be used as an antagonist.

[0090]By using immunogens derived from an ActRIIa polypeptide or an
activin polypeptide, anti-protein/anti-peptide antisera or monoclonal
antibodies can be made by standard protocols (see, for example,
Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring
Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can
be immunized with an immunogenic form of the activin or ActRIIa
polypeptide, an antigenic fragment which is capable of eliciting an
antibody response, or a fusion protein. Techniques for conferring
immunogenicity on a protein or peptide include conjugation to carriers or
other techniques well known in the art. An immunogenic portion of an
ActRIIa or activin polypeptide can be administered in the presence of
adjuvant. The progress of immunization can be monitored by detection of
antibody titers in plasma or serum. Standard ELISA or other immunoassays
can be used with the immunogen as antigen to assess the levels of
antibodies.

[0091]Following immunization of an animal with an antigenic preparation of
an activin or ActRIIa polypeptide, antisera can be obtained and, if
desired, polyclonal antibodies can be isolated from the serum. To produce
monoclonal antibodies, antibody-producing cells (lymphocytes) can be
harvested from an immunized animal and fused by standard somatic cell
fusion procedures with immortalizing cells such as myeloma cells to yield
hybridoma cells. Such techniques are well known in the art, and include,
for example, the hybridoma technique (originally developed by Kohler and
Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma
technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the
EBV-hybridoma technique to produce human monoclonal antibodies (Cole et
al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
pp. 77-96). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with an activin or ActRIIa
polypeptide and monoclonal antibodies isolated from a culture comprising
such hybridoma cells.

[0092]The term "antibody" as used herein is intended to include whole
antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes
fragments or domains of immunoglobulins which are reactive with a
selected antigen. Antibodies can be fragmented using conventional
techniques and the fragments screened for utility and/or interaction with
a specific epitope of interest. Thus, the term includes segments of
proteolytically-cleaved or recombinantly-prepared portions of an antibody
molecule that are capable of selectively reacting with a certain protein.
Non-limiting examples of such proteolytic and/or recombinant fragments
include Fab, F(ab')2, Fab', Fv, and single chain antibodies (scFv)
containing a V[L] and/or V[H] domain joined by a peptide linker. The
scFv's may be covalently or non-covalently linked to form antibodies
having two or more binding sites. The term antibody also includes
polyclonal, monoclonal, or other purified preparations of antibodies and
recombinant antibodies. The term "recombinant antibody", means an
antibody, or antigen binding domain of an immunoglobulin, expressed from
a nucleic acid that has been constructed using the techniques of
molecular biology, such as a humanized antibody or a fully human antibody
developed from a single chain antibody. Single domain and single chain
antibodies are also included within the term "recombinant antibody".

[0093]In certain embodiments, an antibody of the invention is a monoclonal
antibody, and in certain embodiments, the invention makes available
methods for generating novel antibodies. For example, a method for
generating a monoclonal antibody that binds specifically to an ActRIIa
polypeptide or activin polypeptide may comprise administering to a mouse
an amount of an immunogenic composition comprising the antigen
polypeptide effective to stimulate a detectable immune response,
obtaining antibody-producing cells (e.g., cells from the spleen) from the
mouse and fusing the antibody-producing cells with myeloma cells to
obtain antibody-producing hybridomas, and testing the antibody-producing
hybridomas to identify a hybridoma that produces a monocolonal antibody
that binds specifically to the antigen. Once obtained, a hybridoma can be
propagated in a cell culture, optionally in culture conditions where the
hybridoma-derived cells produce the monoclonal antibody that binds
specifically to the antigen. The monoclonal antibody may be purified from
the cell culture.

[0094]The adjective "specifically reactive with" as used in reference to
an antibody is intended to mean, as is generally understood in the art,
that the antibody is sufficiently selective between the antigen of
interest (e.g., an activin or ActRIIa polypeptide) and other antigens
that are not of interest that the antibody is useful for, at minimum,
detecting the presence of the antigen of interest in a particular type of
biological sample. In certain methods employing the antibody, such as
therapeutic applications, a higher degree of specificity in binding may
be desirable. Monoclonal antibodies generally have a greater tendency (as
compared to polyclonal antibodies) to discriminate effectively between
the desired antigens and cross-reacting polypeptides. One characteristic
that influences the specificity of an antibody:antigen interaction is the
affinity of the antibody for the antigen. Although the desired
specificity may be reached with a range of different affinities,
generally preferred antibodies will have an affinity (a dissociation
constant) of about 10-6, 10-7, 10-8, 10-9 M or less.

[0095]In addition, the techniques used to screen antibodies in order to
identify a desirable antibody may influence the properties of the
antibody obtained. For example, if an antibody is to be used for binding
an antigen in solution, it may be desirable to test solution binding. A
variety of different techniques are available for testing interaction
between antibodies and antigens to identify particularly desirable
antibodies. Such techniques include ELISAs, surface plasmon resonance
binding assays (e.g., the Biacore® binding assay, Biacore AB, Uppsala,
Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN
International, Inc., Gaithersburg, Md.), western blots,
immunoprecipitation assays, and immunohistochemistry.

[0096]Examples of categories of nucleic acid compounds that are activin or
ActRIIa antagonists include antisense nucleic acids, RNAi constructs and
catalytic nucleic acid constructs. A nucleic acid compound may be single
or double stranded. A double stranded compound may also include regions
of overhang or non-complementarity, where one or the other of the strands
is single stranded. A single stranded compound may include regions of
self-complementarity, meaning that the compound forms a so-called
"hairpin" or "stem-loop" structure, with a region of double helical
structure. A nucleic acid compound may comprise a nucleotide sequence
that is complementary to a region consisting of no more than 1000, no
more than 500, no more than 250, no more than 100, or no more than 50,
35, 25, 22, 20, 18 or 15 nucleotides of the full-length ActRIIa nucleic
acid sequence or activin βA, βB, βC, or
βE nucleic acid sequence. The region of complementarity will
preferably be at least 8 nucleotides, and optionally about 18 to 35
nucleotides. A region of complementarity may fall within an intron, a
coding sequence or a noncoding sequence of the target transcript, such as
the coding sequence portion. Generally, a nucleic acid compound will have
a length of about 8 to about 500 nucleotides or base pairs in length, and
optionally the length will be about 14 to about 50 nucleotides. A nucleic
acid may be a DNA (particularly for use as an antisense), RNA or RNA:DNA
hybrid. Any one strand may include a mixture of DNA and RNA, as well as
modified forms that cannot readily be classified as either DNA or RNA.
Likewise, a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA,
and any one strand may also include a mixture of DNA and RNA, as well as
modified forms that cannot readily be classified as either DNA or RNA. A
nucleic acid compound may include any of a variety of modifications,
including one or modifications to the backbone (the sugar-phosphate
portion in a natural nucleic acid, including intemucleotide linkages) or
the base portion (the purine or pyrimidine portion of a natural nucleic
acid). An antisense nucleic acid compound will preferably have a length
of about 15 to about 30 nucleotides and will often contain one or more
modifications to improve characteristics such as stability in the serum,
in a cell or in a place where the compound is likely to be delivered,
such as the stomach in the case of orally delivered compounds and the
lung for inhaled compounds. In the case of an RNAi construct, the strand
complementary to the target transcript will generally be RNA or
modifications thereof. The other strand may be RNA, DNA or any other
variation. The duplex portion of double stranded or single stranded
"hairpin" RNAi construct will generally have a length of 18 to 40
nucleotides in length and optionally about 21 to 23 nucleotides in
length, so long as it serves as a Dicer substrate. Catalytic or enzymatic
nucleic acids may be ribozymes or DNA enzymes and may also contain
modified forms. Nucleic acid compounds may inhibit expression of the
target by about 50%, 75%, 90% or more when contacted with cells under
physiological conditions and at a concentration where a nonsense or sense
control has little or no effect. Preferred concentrations for testing the
effect of nucleic acid compounds are 1, 5 and 10 micromolar. Nucleic acid
compounds may also be tested for effects on, for example, red blood cell
levels.

[0097]In certain embodiments, an activin-ActRIIa antagonist may be a
follistatin polypeptide that antagonizes activin bioactivity and/or binds
to activin. The term "follistatin polypeptide" includes polypeptides
comprising any naturally occurring polypeptide of follistatin as well as
any variants thereof (including mutants, fragments, fusions, and
peptidomimetic forms) that retain a useful activity, and further includes
any functional monomer or multimer of follistatin. Variants of
follistatin polypeptides that retain activin binding properties can be
identified based on previous studies involving follistatin and activin
interactions. For example, WO2008/030367 discloses specific follistatin
domains ("FSDs") that are shown to be important for activin binding. As
shown below in SEQ ID NOs: 19-21, the N-terminus follistatin domain
("FSND" SEQ ID NO: 19), FSD2 (SEQ ID NO: 20), and to a lesser extent FSD1
(SEQ ID NO: 21) represent exemplary domains within follistatin important
for activin binding. In addition, methods for making and testing
libraries of polypeptides are described above in the context of ActRIIa
polypeptides and such methods also pertain to making and testing variants
of follistatin. Follistatin polypeptides include polypeptides derived
from the sequence of any known follistatin having a sequence at least
about 80% identical to the sequence of a follistatin polypeptide, and
optionally at least 85%, 90%, 95%, 97%, 99% or greater identity. Examples
of follistatin polypeptides include the mature follistatin polypeptide or
shorter isoforms or other variants of the human follistatin precursor
polypeptide (SEQ ID NO: 17) as described, for example, in WO2005/025601.

[0102]In other embodiments, an activin-ActRIIa antagonist may be a
follistatin-like related gene (FLRG) that antagonizes activin bioactivity
and/or binds to activin. The term "FLRG polypeptide" includes
polypeptides comprising any naturally occurring polypeptide of FLRG as
well as any variants thereof (including mutants, fragments, fusions, and
peptidomimetic forms) that retain a useful activity. Variants of FLRG
polypeptides that retain activin binding properties can be identified
using routine methods to assay FLRG and activin interactions. See, for
example, U.S. Pat. No. 6,537,966. In addition, methods for making and
testing libraries of polypeptides are described above in the context of
ActRIIa polypeptides and such methods also pertain to making and testing
variants of FLRG. FLRG polypeptides include polypeptides derived from the
sequence of any known FLRG having a sequence at least about 80% identical
to the sequence of an FLRG polypeptide, and optionally at least 85%, 90%,
95%, 97%, 99% or greater identity.

[0104]In certain embodiments, functional variants or modified forms of the
follistatin polypeptides and FLRG polypeptides include fusion protein
having at least a portion of the follistatin polypeptides or FLRG
polypeptides and one or more fusion domains, such as, for example,
domains that facilitate isolation, detection, stabilization or
multimerization of the polypeptide. Suitable fusion domains are discussed
in detail above with reference to the ActRIIa polypeptides. In one
embodiment, an activin-ActRIIa antagonist is a fusion protein comprising
an activin binding portion of a follistaton polypeptide fused to an Fc
domain. In another embodiment, an activin-ActRIIa antagonist is a fusion
protein comprising an activin binding portion of an FLRG polypeptide
fused to an Fc domain. Follistatin and FLRG have been shown in the
literature, and by the applicants with respect to FLRG, to have
affinities for Activin A in the picomolar range, indicating that these
agents will inhibit activin A signaling to a similar degree as
ActRIIa-Fc.

5. Exemplary Therapeutic Methods

[0105]In certain embodiments, the present invention provides methods for
managing a patient that has been treated with, or is a candidate to be
treated with, an activin-ActRIIa antagonist by measuring one or more
hematologic parameters in the patient. The hematologic parameters may be
used to evaluate appropriate dosing for a patient who is a candidate to
be treated with an activin-ActRIIa anatagonist, to monitor the
hematologic parameters during treatment with an activin-ActRIIa
antagonist, to evaluate whether to adjust the dosage during treatment
with an activin-ActRIIa antagonist, and/or to evaluate an appropriate
maintenance dose of an activin-ActRIIa antagonist. If one or more of the
hematologic parameters are outside the normal level, dosing with the
activin-ActRIIa antagonist may be reduced, delayed or terminated.

[0106]Hematologic parameters that may be measured in accordance with the
methods provided herein include, for example, red blood cell levels,
blood pressure, iron stores, and other agents found in bodily fluids that
correlate with increased red blood cell levels, using art recognized
methods. Such parameters may be determined using a blood sample from a
patient. Increases in red blood cell levels, hemoglobin levels, and/or
hematocrit levels may cause increases in blood pressure.

[0107]Red blood cell levels may be determined, for example, by determining
a red blood cell count, by measuring hemoglobin levels or by measuring
hematocrit levels. A red blood cell count may be determined using a
commercially available Coulter Counter. The normal range for a red blood
cell count is between 4.2-5.9 million cells/cm, although individual
variations should be taken into account. Hemoglobin levels may be
determined by lysing the red blood cells, converting the hemoglobin into
cyanomethemoglobin and measuring the amount of hemoglobin with a
calorimeter. The normal ranges for hemoglobin are 14-18 gm/dl for adult
males and 12-16 gm-dl for adult females, although individual variations
should be taken into account. Hematocrit (Hct) or packed cell volume
(PCV) refers to the ratio of the volume of red blood cells to the volume
of whole blood. Hematocrit may be determined, for example, by
centrifugation of a blood sample followed by analysis of the layers
produced. Normal ranges for hematocrit are approximately 41-51% for men
and 35-45% for women, although individual variations should be taken into
account.

[0108]Blood pressure, including systolic blood pressure, diastolic blood
pressure, or mean arterial blood pressure, may be determined using art
recognized methods. Blood pressure is most commonly measured using a
sphygmomanometer. Typical values for a resting, healthy adult human are
approximately 120 mmHg systolic and 80 mmHg diastolic, although
individual variations should be taken into account. Individuals suffering
from hypertension typically have a blood pressure ≧140 mmHg
systolic and ≧90 diastolic blood pressure. Individuals having a
level above normal but less than 140/90 mmHg are generally referred to as
prehypertensive. Additional methods for measuring blood pressure may be
found in Pickering et al., Hypertension 45: 142-161 (2005).

[0109]Iron stores may be measured using a variety of art recognized
techniques including, for example, by determining levels of one or more
of the following: serum ferritin (SF), transferrin saturation (TSAT),
total iron binding capacity, hemoglobin concentration, zinc
protoporphyrin, mean cell volume (MCV), or transferrin receptor in serum.
Serum ferritin levels may be determined, for example, using an
immunoassay such as an enzyme-linked immunosorbent assay (ELISA) or
immunoturbidometry. In normal patients, serum ferritin levels range from
13 to 220 ng/mL, although individual variations should be taken into
account. Transferrin saturation levels represent the proportion of
transferrin bound to iron and may be determined by dividing serum iron by
total iron biding capacity (TIBC). In normal patients, transferrin
saturation levels range from 20% to 40%, although individual variations
should be taken into account. Serum iron may be determiend using
colorimetry and is expressed as ug/dl or umol/l. Total iron binding
capacity reflects the total capacity of circulating transferrin bound to
iron and may be determined using a colorimetric assay to determine the
amount of iron that can be bound to unsaturated transferrin in vitro. The
normal range of total iron binding capacity is about 250-450 ug/dl,
although individual variations should be taken into account. Additional
information about measuring iron stores may be found in the World Health
Organization report entitled Assessing the Iron Status of Populations
dated April 2004 and in Yamanishi et al., Clinical Chemistry 48:
1565-1570 (2002).

[0110]In one embodiment, if one or more hematologic parameters are outside
the normal range, or on the high side of normal, in a patient who is a
candidate to be treated with an activin-ActRIIa antagonist then onset of
administration of the activin-ActRIIa antagonist may be delayed until the
hematologic parameters have returned to a normal or acceptable level
either naturally or via therapeutic intervention. For example, if a
candidate patient is hypertensive or prehypertensive, then the patient
may be treated with a blood pressure lowering agent in order to reduce
the patient's blood pressure. Any blood pressure lowering agent
appropriate for the individual patient's condition may be used including,
for example, diuretics, adrenergic inhibitors (including alpha blockers
and beta blockers), vasodilators, calcium channel blockers,
angiotensin-converting enzyme (ACE) inhibitors, or angiotensin II
receptor blockers. Blood pressure may alternatively be treated using a
diet and exercise regimen. Similarly, if a candidate patient has iron
stores that are lower than normal, or on the low side of normal, then the
patient may be treated with an appropriate regimen of diet and/or iron
supplements until the patient's iron stores have returned to a normal or
acceptable level. For patients having higher than normal red blood cell
levels and/or hemoglobin levels, then administration of the
activin-ActRIIa antagonist may be delayed until the levels have returned
to a normal or acceptable level.

[0111]In certain embodiments, if one or more hematologic parameters are
outside the normal range, or on the high side of normal, in a patient who
is a candidate to be treated with an activin-ActRIIa antagonist then the
onset of administration may be not be delayed. However, the dosage amount
or frequency of dosing of the activin-ActRIIa antagonist may be set at an
amount that would reduce the risk of an unacceptable increase in the
hematologic parameters arising upon administration of the activin-ActRIIa
antagonist. Alternatively, a therapeutic regimen may be developed for the
patient that combines an activin-ActRIIa antagonist with a therapeutic
agent that addresses the undesirable level of the hematologic parameter.
For example, if the patient has elevated blood pressure, then a
therapeutic regimen involving administration of an activin-ActRIIa
antagonist and a blood pressure lowering agent may be designed. For a
patient having lower than desired iron stores, a therapeutic regimen of
an activin-ActRIIa antagonist and iron supplementation may be developed.

[0112]In one embodiment, baseline parameter(s) for one or more hematologic
parameters may be established for a patient who is a candidate to be
treated with an activin-ActRIIa antagonists and an appropriate dosing
regimen establish for that patient based on the baseline value(s).
Alternatively, established baseline parameters based on a patient's
medical history could be used to inform an appropriate activin-ActRIIa
antagonist dosing regimen for a patient. For example, if a healthy
patient has an established baseline blood pressure reading that is above
the defined normal range it may not be necessary to bring the patient's
blood pressure into the range that is considered normal for the general
population prior to treatment with the activin-ActRIIa antagonist. A
patient's baseline values for one or more hematologic parameters prior to
treatment with an activin-ActRIIa antagonist may also be used as the
relevant comparative values for monitoring any changes to the hematologic
parameters during treatment with the activin-ActRIIa antagonist.

[0113]In certain embodiments, one or more hematologic parameters are
measured in patients who are being treated with an activin-ActRIIa
antagonist. The hematologic parameters may be used to monitor the patient
during treatment and permit adjustment or termination of the dosing with
the activin-ActRIIa antagonist or additional dosing with another
therapeutic agent. For example, if administration of an activin-ActRIIa
antagonist results in an increase in blood pressure, red blood cell
level, or hemoglobin level, or a reduction in iron stores, then the dose
of the activin-ActRIIa antagonists may be reduced in amount or frequency
in order to decrease the effects of the activin-ActRIIa antagonist on the
one or more hematologic parameters. If administration or an
activin-ActRIIa antagonist results in a change in one or more hematologic
parameters that is adverse to the patient, then the dosing of the
activin-ActRIIa antagonist may be terminated either temporarily, until
the hematologic parameter(s) return to an acceptable level, or
permanently. Similarly, if one or more hematologic parameters are not
brought within an acceptable range after reducing the dose or frequency
of administration of the activin-ActRIIa antagonist then the dosing may
be terminated. As an alternative, or in addition to, reducing or
terminating the dosing with the activin-ActRIIa antagonist, the patient
may be dosed with an additional therapeutic agent that addresses the
undesirable level in the hematologic parameter(s), such as, for example,
a blood pressure lowering agent or an iron supplement. For example, if a
patient being treated with an activin-ActRIIa antagonist has elevated
blood pressure, then dosing with the activin-ActRIIa antagonist may
continue at the same level and a blood pressure lowering agent is added
to the treatment regimen, dosing with the activin-ActRIIa antagonist may
be reduce (e.g., in amount and/or frequency) and a blood pressure
lowering agent is added to the treatment regimen, or dosing with the
activin-ActRIIa antagonist may be terminated and the patient may be
treated with a blood pressure lowering agent.

[0114]In certain embodiments, if a patient being treated with an
activin-ActRIIa antagonist or a patient who is a candidate for treatment
with an activin-ActRIIa antagonist has one or more of the following: a
hemoglobin level greater than 12 g/dl, a hemoglobin level greater than 15
g/dl, a blood pressure ≧120/80 mmHg, a blood pressure
≧140/90 mmHg, a transferrin saturation level less than 20%, and/or
a ferritin level less than 100 ng/ml, then dosing with the
activin-ActRIIa antagonist is reduced, delayed or terminated. As an
alternative, or in addition to, reducing, delaying or terminating dosing
with activin-ActRIIa antagonist, a therapeutic agent that addresses the
undesired level of one or more hematologic parameters (such as a blood
pressure lowering agent or an iron supplement) may be administered to the
patient.

[0115]In one embodiment, the present invention provides a method for
dosing a patient with an activin-ActRIIa antagonist by administering to
the patient an activin-ActRIIa antagonist in an amount and at a frequency
which reduces the risk of causing a rise in hemoglobin levels greater
than 1 g/dl over a two week period. The methods may comprise measuring
one or more hematologic parameters either before beginning administration
of the activin-ActRIIa antagonist and/or during administration of the
activin-ActRIIa antagonist. The initial dose of the activin-ActRIIa
antagonist may be set so as to reduce the risk of causing a rise in
hemoglobin levels greater than 1 g/dl over a two week period. In
addition, the dose may be adjusted over time to in order to maintain a
reduced risk of causing a rise in hemoglobin levels greater than 1 g/dl
in two weeks.

[0116]In certain embodiments, the present invention provides a method for
administering an ActRIIa-Fc fusion protein to a patient by administering
the ActRIIa fusion protein no more frequently than once per 60 days, once
per 90 days, or once per 120 days, or 1-6 times per year, 2-6 times per
year, 1-5 times per year, 2-5 times per year, 1-4 times per year, 2-4
times per year, 1-3 times per year, or 2-3 times per year. As
demonstrated herein, increases in red blood cell levels arising from
administration peak around 60 days after administration. At about 90 days
after administration, a significant reduction in red blood cell levels is
seen and after about 120 days red blood cell levels return to the
baseline level. Accordingly, for patients in which the activin-ActRIIa
antagonist is being administered for purposes other than increasing red
blood cell levels, it may be desirable to administer subsequent doses of
the activin-ActRIIa antagonist after the peak increase in red blood cell
levels from the previous dose, or even after red blood cell levels have
returned to normal.

[0117]In certain embodiments, the invention provides methods for
determining dosing and monitoring therapeutic progress with
activin-ActRIIa antagonist treatment in patients in which the
activin-ActRIIa antagonist is being administered to increase red blood
cell levels. The methods involve determining one or more hematologic
parameters either prior to beginning dosing with the activin-ActRIIa
antagonist and/or during treatment with the activin-ActRIIa antagonist.
For example, one or more hematologic parameters may be determined in a
patient who is a candidate for administration of an activin-ActRIIa
antagonist for increasing blood cell levels to facilitate determination
of dosage amount and frequency. One or more hematologic parameters may
also be determined in a patient being treated with an activin-ActRIIa
antagonist for purposes of increasing red blood cell levels in order to
monitor progress of the treatment, facilitate dosing adjustments, and to
determine maintenance dosing levels, etc.

[0118]In accordance with the methods of the invention, one or more
hematologic parameters may be measured at various time points and at
varying frequencies as needed for an individual patient based on various
factors such as a patient's baseline levels, responsiveness to treatment
with an activin-ActRIIa antagonist, general health, age, sex, weight,
etc. Measuring of one or more hematologic parameters may be carried out
before and/or during treatment with an activin-ActRIIa antagonist. If
conducting multiple measurements of hematologic parameters at various
time points, the same set of hematologic parameter(s) need not be
measured at each time point. Similarly, the same test for an individual
parameter need not be used at each time point. Appropriate hematologic
parameters and tests for such parameters may be chosen for an individual
taking into account factors specific to the given individual. Testing of
hematologic parameters may occur as frequently as needed for a given
individual, such as, for example, once per day, once per week, once per
every two weeks, once per month, once per each 2 month period, once per
each 3 month period, once per each 6 month period, or once per year. In
addition, the frequency of testing may vary over time. For example, upon
initial dosing of an individual it may be desirable to test for one or
more hematologic parameters more frequently, e.g., once per day, once per
week, once per every two weeks, or once per month, and then decrease the
frequency of testing over time, e.g., after one month, two months, six
months, 1 year, two years, or longer, of treatment, the frequency of
testing may reduced to, for example, once per month, once per every two
months, once per every three months, once per every six months, or once
per year. Similarly, it may be desirable to test more frequently when
adjusting a patient's dose of an activin-ActRIIa antagonist (e.g., either
amount or frequency of administration) and then decrease the frequency of
testing over time, for example, once the patient's response to the
activin-ActRIIa antagonist has been established.

[0119]In various embodiments, patients being treated with an
activin-ActRIIa antagonist, or candidate patients for treatment with an
activin-ActRIIa antagonist, may be mammals such as rodents and primates,
and particularly human patients.

[0121]In certain embodiments, patients being treated with an
activin-ActRIIa antagonist, or candidate patients to be treated with an
activin-ActRIIa antagonist, are patients suffering from or at high risk
for developing breast cancer. As every woman is at risk for developing
breast cancer, a woman with a high risk for developing breast cancer is a
woman whose risk factors confer a greater probability of developing the
disease compared to the general population or the population of women
within a certain age group. Exemplary risk factors include age, family
history or genetic makeup, lifestyle habits such as exercise and diet,
exposure to radiation or other cancer-causing agents, age at the time the
first child was born, genetic changes, and weight gain after menopause.
Exemplary patients include, for example, patients that have mutations in
the BRCA1/2 genes or other genes shown to predispose women to breast and
ovarian cancer are also included. Patients also include individuals with
solid tumors, metastatic cancer, precancerous lesions of the breast,
benign lesions of the breast, or with any abnormal proliferative lesions
including typical hyperplasia, atypical hyperplasia, and noninvasive or
in situ carcinoma. Patients also include those with both
hormone-dependent or hormone-responsive cancers (e.g., estrogen receptor
positive cancers) and hormone-independent cancers (e.g., estrogen
receptor negative or estrogen receptor mutant cancers). Patients
suffering from cancers in which growth factors or oncogenes are activated
(e.g., breast cancers in which c-erbB-2 (also known as HER-2/Neu)
tyrosine kinase is expressed) are also contemplated.

[0122]In certain embodiments, patients being treated with an
activin-ActRIIa antagonist, or candidate patients to be treated with an
activin-ActRIIa antagonist, are patients with undesirably low red blood
cell or hemoglobin levels, such as patients having an anemia, and those
that are at risk for developing undesirably low red blood cell or
hemoglobin levels, such as those patients that are about to undergo major
surgery or other procedures that may result in substantial blood loss,
such as having blood drawn and stored for a later transfusion. Patients
and candidate patients may also include those patients in need of an
increase in red blood cells and/or hemoglobin levels that do not respond
well to Epo. When observing hemoglobin levels in humans, a level of less
than normal for the appropriate age and gender category may be indicative
of anemia, although individual variations are taken into account.
Potential causes of anemia include blood-loss, nutritional deficits,
medication reaction, various problems with the bone marrow and many
diseases. More particularly, anemia has been associated with a variety of
disorders that include, for example, chronic renal failure,
myelodysplastic syndrome, rheumatoid arthritis, bone marrow
transplantation. Anemia may also be associated with the following
conditions: solid tumors (e.g. breast cancer, lung cancer, colon cancer);
tumors of the lymphatic system (e.g. chronic lymphocyte leukemia,
non-Hodgkins and Hodgkins lymphomas); tumors of the hematopoietic system
(eg. leukemia, myelodysplastic syndrome, multiple myeloma); radiation
therapy; chemotherapy (e.g. platinum containing regimens); inflammatory
and autoimmune diseases, including, but not limited to, rheumatoid
arthritis, other inflammatory arthritides, systemic lupus erythematosis
(SLE), acute or chronic skin diseases (e.g. psoriasis), inflammatory
bowel disease (e.g. Crohn's disease and ulcerative colitis); acute or
chronic renal disease or failure including idiopathic or congenital
conditions; acute or chronic liver disease; acute or chronic bleeding;
situations where transfusion of red blood cells is not possible due to
patient allo- or auto-antibodies and/or for religious reasons (e.g. some
Jehovah's Witnesses); infections (e.g. malaria, osteomyelitis);
hemoglobinopathies, including, for example, sickle cell disease,
thalassemias; drug use or abuse, e.g. alcohol misuse; pediatric patients
with anemia from any cause to avoid transfusion; and elderly patients or
patients with underlying cardiopulmonary disease with anemia who cannot
receive transfusions due to concerns about circulatory overload.

[0123]As used herein, a therapeutic that "prevents" a disorder or
condition refers to a compound that, in a statistical sample, reduces the
occurrence of the disorder or condition in the treated sample relative to
an untreated control sample, or delays the onset or reduces the severity
of one or more symptoms of the disorder or condition relative to the
untreated control sample. The term "treating" as used herein includes
prophylaxis of the named condition or amelioration or elimination of the
condition once it has been established. In either case, prevention or
treatment may be discerned in the diagnosis provided by a physician or
other health care provider and the intended result of administration of
the therapeutic agent.

6. Pharmaceutical Compositions

[0124]In certain embodiments, activin-ActRIIa antagonists (e.g., ActRIIa
polypeptides) of the present invention are formulated with a
pharmaceutically acceptable carrier. For example, an ActRIIa polypeptide
can be administered alone or as a component of a pharmaceutical
formulation (therapeutic composition). The subject compounds may be
formulated for administration in any convenient way for use in human or
veterinary medicine.

[0125]In certain embodiments, the therapeutic method of the invention
includes administering the composition systemically, or locally as an
implant or device. When administered, the therapeutic composition for use
in this invention is, of course, in a pyrogen-free, physiologically
acceptable form. Therapeutically useful agents other than the
activin-ActRIIa antagonists which may also optionally be included in the
composition as described above, may be administered simultaneously or
sequentially with the subject compounds (e.g., ActRIIa polypeptides) in
the methods of the invention.

[0126]Typically, activin-ActRIaI antagonists will be administered
parenterally. Pharmaceutical compositions suitable for parenteral
administration may comprise one or more ActRIIa polypeptides in
combination with one or more pharmaceutically acceptable sterile isotonic
aqueous or nonaqueous solutions, dispersions, suspensions or emulsions,
or sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents. Examples of suitable aqueous and
nonaqueous carriers which may be employed in the pharmaceutical
compositions of the invention include water, ethanol, polyols (such as
glycerol, propylene glycol, polyethylene glycol, and the like), and
suitable mixtures thereof, vegetable oils, such as olive oil, and
injectable organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as
lecithin, by the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.

[0127]Further, the composition may be encapsulated or injected in a form
for delivery to a target tissue site (e.g., bone marrow). In certain
embodiments, compositions of the present invention may include a matrix
capable of delivering one or more therapeutic compounds (e.g., ActRIIa
polypeptides) to a target tissue site (e.g., bone marrow), providing a
structure for the developing tissue and optimally capable of being
resorbed into the body. For example, the matrix may provide slow release
of the ActRIIa polypeptides. Such matrices may be formed of materials
presently in use for other implanted medical applications.

[0128]The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the subject
compositions will define the appropriate formulation. Potential matrices
for the compositions may be biodegradable and chemically defined calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and
polyanhydrides. Other potential materials are biodegradable and
biologically well defined, such as bone or dermal collagen. Further
matrices are comprised of pure proteins or extracellular matrix
components. Other potential matrices are non-biodegradable and chemically
defined, such as sintered hydroxyapatite, bioglass, aluminates, or other
ceramics. Matrices may be comprised of combinations of any of the above
mentioned types of material, such as polylactic acid and hydroxyapatite
or collagen and tricalciumphosphate. The bioceramics may be altered in
composition, such as in calcium-aluminate-phosphate and processing to
alter pore size, particle size, particle shape, and biodegradability.

[0129]In certain embodiments, methods of the invention can be administered
for orally, e.g., in the form of capsules, cachets, pills, tablets,
lozenges (using a flavored basis, usually sucrose and acacia or
tragacanth), powders, granules, or as a solution or a suspension in an
aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil
liquid emulsion, or as an elixir or syrup, or as pastilles (using an
inert base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined amount of
an agent as an active ingredient. An agent may also be administered as a
bolus, electuary or paste.

[0130]In solid dosage forms for oral administration (capsules, tablets,
pills, dragees, powders, granules, and the like), one or more therapeutic
compounds of the present invention may be mixed with one or more
pharmaceutically acceptable carriers, such as sodium citrate or dicalcium
phosphate, and/or any of the following: (1) fillers or extenders, such as
starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2)
binders, such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants,
such as glycerol; (4) disintegrating agents, such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay; (9)
lubricants, such a talc, calcium stearate, magnesium stearate, solid
polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and
(10) coloring agents. In the case of capsules, tablets and pills, the
pharmaceutical compositions may also comprise buffering agents. Solid
compositions of a similar type may also be employed as fillers in soft
and hard-filled gelatin capsules using such excipients as lactose or milk
sugars, as well as high molecular weight polyethylene glycols and the
like.

[0133]The compositions of the invention may also contain adjuvants, such
as preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured by the
inclusion of various antibacterial and antifungal agents, for example,
paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium chloride,
and the like into the compositions. In addition, prolonged absorption of
the injectable pharmaceutical form may be brought about by the inclusion
of agents which delay absorption, such as aluminum monostearate and
gelatin.

[0134]It is understood that the dosage regimen will be determined by the
attending physician considering various factors which modify the action
of the subject compounds of the invention (e.g., ActRIIa polypeptides).
The various factors include, but are not limited to, the patient's red
blood cell count, hemoglobin level or other diagnostic assessments, the
desired target red blood cell count, the patient's age, sex, and diet,
the severity of any disease that may be contributing to a depressed red
blood cell level, time of administration, and other clinical factors. The
addition of other known growth factors to the final composition may also
affect the dosage. Progress can be monitored by periodic assessment of
red blood cell and hemoglobin levels, as well as assessments of
reticulocyte levels and other indicators of the hematopoietic process.

[0135]Experiments with primates and humans have demonstrated that effects
of ActRIIa-Fc on red blood cell levels are detectable when the compound
is dosed at intervals and amounts sufficient to achieve serum
concentrations of about 100 ng/ml or greater, for a period of at least
about 20 to 30 days. Dosing to obtain serum levels of 200 ng/ml, 500
ng/ml, 1000 ng/ml or greater for a period of at least 20 to 30 days may
also be used. Bone effects can be observed at serum levels of about 200
ng/ml, with substantial effects beginning at about 1000 ng/ml or higher,
over a period of at least about 20 to 30 days. Thus, if it is desirable
to achieve effects on red blood cells while having little effect on bone,
a dosing scheme may be designed to deliver a serum concentration of
between about 100 and 1000 ng/ml over a period of about 20 to 30 days.
Alternatively, if it is desirable to achieve effects on bone, breast
cancer, etc., while having little effect on, or reducing effects on red
blood cell levels, a dosing scheme may be designed to deliver a dosing
scheme of between about 100 and 1000 ng/ml with a dosing frequency that
occurs less than once every 60 days, once every 90 days, or once every
120 days. In humans, serum levels of 200 ng/ml may be achieved with a
single dose of 0.1 mg/kg or greater and serum levels of 1000 ng/ml may be
achieved with a single dose of 0.3 mg/kg or greater. The observed serum
half-life of the molecule is between about 20 and 30 days, substantially
longer than most Fc fusion proteins, and thus a sustained effective serum
level may be achieved, for example, by dosing with about 0.05 to 0.5
mg/kg on a weekly or biweekly basis, or higher doses may be used with
longer intervals between dosings. For example, doses of 0.1 to 1 mg/kg
might be used on a monthly or bimonthly basis.

[0136]In certain embodiments, the present invention also provides gene
therapy for the in vivo production of ActRIIa polypeptides. Such therapy
would achieve its therapeutic effect by introduction of the ActRIIa
polynucleotide sequences into cells or tissues having the disorders as
listed above. Delivery of ActRIIa polynucleotide sequences can be
achieved using a recombinant expression vector such as a chimeric virus
or a colloidal dispersion system. Preferred for therapeutic delivery of
ActRIIa polynucleotide sequences is the use of targeted liposomes.

[0137]Various viral vectors which can be utilized for gene therapy as
taught herein include adenovirus, herpes virus, vaccinia, or an RNA virus
such as a retrovirus. The retroviral vector may be a derivative of a
murine or avian retrovirus. Examples of retroviral vectors in which a
single foreign gene can be inserted include, but are not limited to:
Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus
(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus
(RSV). A number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for a
selectable marker so that transduced cells can be identified and
generated. Retroviral vectors can be made target-specific by attaching,
for example, a sugar, a glycolipid, or a protein. Preferred targeting is
accomplished by using an antibody. Those of skill in the art will
recognize that specific polynucleotide sequences can be inserted into the
retroviral genome or attached to a viral envelope to allow target
specific delivery of the retroviral vector containing the ActRIIa
polynucleotide.

[0138]Alternatively, tissue culture cells can be directly transfected with
plasmids encoding the retroviral structural genes gag, pol and env, by
conventional calcium phosphate transfection. These cells are then
transfected with the vector plasmid containing the genes of interest. The
resulting cells release the retroviral vector into the culture medium.

[0139]Another targeted delivery system for ActRIIa polynucleotides is a
colloidal dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles, mixed
micelles, and liposomes. The preferred colloidal system of this invention
is a liposome. Liposomes are artificial membrane vesicles which are
useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact
virions can be encapsulated within the aqueous interior and be delivered
to cells in a biologically active form (see e.g., Fraley, et al., Trends
Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using a
liposome vehicle, are known in the art, see e.g., Mannino, et al.,
Biotechniques, 6:682, 1988. The composition of the liposome is usually a
combination of phospholipids, usually in combination with steroids,
especially cholesterol. Other phospholipids or other lipids may also be
used. The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.

[0140]Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Illustrative phospholipids
include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and
distearoylphosphatidylcholine. The targeting of liposomes is also
possible based on, for example, organ-specificity, cell-specificity, and
organelle-specificity and is known in the art.

Ememplification

[0141]The invention now being generally described, it will be more readily
understood by reference to the following examples, which are included
merely for purposes of illustration of certain embodiments and
embodiments of the present invention, and are not intended to limit the
invention.

Example 1

ActRIIa-Fc Fusion Proteins

[0142]Applicants constructed a soluble ActRIIa fusion protein that has the
extracellular domain of human ActRIIa fused to a human or mouse Fc domain
with a minimal linker in between. The constructs are referred to as
ActRIIa-hFc and ActRIIa-mFc, respectively.

[0147]Both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to
recombinant expression. As shown in FIG. 1, the protein was purified as a
single, well-defined peak of protein. N-terminal sequencing revealed a
single sequence of -ILGRSTQE (SEQ ID NO: 11). Purification could be
achieved by a series of column chromatography steps, including, for
example, three or more of the following, in any order: protein A
chromatography, Q sepharose chromatography, phenylsepharose
chromatography, size exclusion chromatography, and cation exchange
chromatography. The purification could be completed with viral filtration
and buffer exchange. The ActRIIa-hFc protein was purified to a purity of
>98% as determined by size exclusion chromatography and >95% as
determined by SDS PAGE.

[0148]ActRIIa-hFc and ActRIIa-mFc showed a high affinity for ligands,
particularly activin A. GDF-11 or Activin A ("ActA") were immobilized on
a Biacore CM5 chip using standard amine coupling procedure. ActRIIa-hFc
and ActRIIa-mFc proteins were loaded onto the system, and binding was
measured. ActRIIa-hFc bound to activin with a dissociation constant (KD)
of 5×10-12, and the protein bound to GDF11 with a KD of
9.96×10-9. See FIG. 2. ActRIIa-mFc behaved similarly.

[0149]The ActRIIa-hFc was very stable in pharmacokinetic studies. Rats
were dosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc protein and
plasma levels of the protein were measured at 24, 48, 72, 144 and 168
hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg or 30
mg/kg. In rats, ActRIIa-hFc had an 11-14 day serum half life and
circulating levels of the drug were quite high after two weeks (11
μg/ml, 110 μg/ml or 304 μg/ml for initial administrations of 1
mg/kg, 10 mg/kg or 30 mg/kg, respectively.) In cynomolgus monkeys, the
plasma half life was substantially greater than 14 days and circulating
levels of the drug were 25 μg/ml, 304 μg/ml or 1440 μg/ml for
initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.
Preliminary results in humans suggests that the serum half life is
between about 20 and 30 days.

Example 2

Characterization of an ActRIIa-hFc Protein

[0150]ActRIIa-hFc fusion protein was expressed in stably transfected
CHO-DUKX B11 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter),
using a tissue plasminogen leader sequence of SEQ ID NO:9. The protein,
purified as described above in Example 1, had a sequence of SEQ ID NO:7.
The Fc portion is a human IgG1 Fc sequence, as shown in SEQ ID NO:7.
Sialic acid analysis showed that the protein contained, on average,
between about 1.5 and 2.5 moles of sialic acid per molecule of
ActRIIa-hFc fusion protein.

[0151]This purified protein showed a remarkably long serum half-life in
all animals tested, including a half-life of 25-32 days in human patients
(see Example 6, below). Additionally, the CHO cell expressed material has
a higher affinity for activin B ligand than that reported for an
ActRIIa-hFc fusion protein expressed in human 293 cells (del Re et al., J
Biol Chem. Dec. 17, 2004;279(51):53126-35.) Additionally, the use of the
tPa leader sequence provided greater production than other leader
sequences and, unlike ActRIIa-Fc expressed with a native leader, provided
a highly pure N-terminal sequence. Use of the native leader sequence
resulted in two major species of ActRIIa-Fc, each having a different
N-terminal sequence.

Example 3

ActRIIa-hFc Increases Red Blood Cell Levels in Non-Human Primates

[0152]The study employed four groups of five male and five female
cynomolgus monkeys each, with three per sex per group scheduled for
termination on Day 29, and two per sex per group scheduled for
termination on Day 57. Each animal was administered the vehicle (Group I)
or ActRIIa-Fc at doses of 1, 10, or 30 mg/kg (Groups 2, 3 and 4,
respectively) via intravenous (IV) injection on Days 1, 8, 15 and 22. The
dose volume was maintained at 3 mL/kg. Various measures of red blood cell
levels were assessed two days prior to the first administration and at
days 15, 29 and 57 (for the remaining two animals) after the first
administration.

[0154]Statistical significance was calculated for each treatment group
relative to the mean for the treatment group at baseline.

[0155]Notably, the increases in red blood cell counts and hemoglobin
levels are roughly equivalent in magnitude to effects reported with
erythropoietin. The onset of these effects is more rapid with ActRIIa-Fc
than with erythropoietin.

[0157]The ActRIIa-hFc fusion protein described in Example 1 was
administered to human patients in a randomized, double-blind,
placebo-controlled study that was conducted to evaluate, primarily, the
safety of the protein in healthy, postmenopausal women. Forty-eight
subjects were randomized in cohorts of 6 to receive either a single dose
of ActRIIa-hFc or placebo (5 active:1 placebo). Dose levels ranged from
0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.1 mg/kg subcutaneously
(SC). All subjects were followed for 120 days. In addition to
pharmacokinetic (PK) analyses, the biologic activity of ActRIIa-hFc was
also assessed by measurement of biochemical markers of bone formation and
resorption, and FSH levels.

[0158]To look for potential changes, hemoglobin and RBC numbers were
examined in detail for all subjects over the course of the study and
compared to the baseline levels. Platelet counts were compared over the
same time as the control. There were no clinically significant changes
from the baseline values over time for the platelet counts.

[0159]PK analysis of ActRIIa-hFc displayed a linear profile with dose, and
a mean half-life of approximately 25-32 days. The area-under-curve (AUC)
for ActRIIa-hFc was linearly related to dose, and the absorption after SC
dosing was essentially complete (see FIGS. 7 and 8). These data indicate
that SC is a desirable approach to dosing because it provides equivalent
bioavailability and serum-half life for the drug while avoiding the spike
in serum concentrations of drug associated with the first few days of IV
dosing (see FIG. 8). ActRIIa-hFc caused a rapid, sustained dose-dependent
increase in serum levels of bone-specific alkaline phosphatase (BAP),
which is a marker for anabolic bone growth, and a dose-dependent decrease
in C-terminal type 1 collagen telopeptide and tartrate-resistant acid
phosphatase 5b levels, which are markers for bone resorption. Other
markers, such as P1NP showed inconclusive results. BAP levels showed near
saturating effects at the highest dosage of drug, indicating that
half-maximal effects on this anabolic bone biomarker could be achieved at
a dosage of 0.3 mg/kg, with increases ranging up to 3 mg/kg. Calculated
as a relationship of pharmacodynamic effect to AUC for drug, the EC50 is
51,465 (day*ng/ml). See FIG. 9. These bone biomarker changes were
sustained for approximately 120 days at the highest dose levels tested.
There was also a dose-dependent decrease in serum FSH levels consistent
with inhibition of activin.

[0160]Overall, there was a very small non-drug related reduction in
hemoglobin over the first week of the study probably related to study
phlebotomy in the 0.01 and 0.03 mg/kg groups whether given IV or SC. The
0.1 mg/kg SC and IV hemoglobin results were stable or showed modest
increases by Day 8-15. At the 0.3 mg/kg IV dose level there was a clear
increase in HGB levels seen as early as Day 2 and often peaking at Day
15-29 that was not seen in the placebo subjects. At the 1.0 mg/kg IV dose
and the 3.0 mg/kg IV dose, mean increases in hemoglobin of greater than 1
g/dl were observed in response to the single dose, with corresponding
increases in RBC counts and hematocrit. These hematologic parameters
peaked at about 60 days after the dose and substantial decrease by day
120. This indicates that dosing for the purpose of increasing red blood
cell levels may be more effective if done at intervals less than 120 days
(i.e., prior to return to baseline), with dosing intervals of 90 days or
less or 60 days or less may be desirable. For a summary of hematological
changes, see FIGS. 10-13.

[0161]Overall, ActRIIa-hFc showed a dose-dependent effect on red blood
cell counts and reticulocyte counts, and a dose-dependent effect on
markers of bone formation.

Example 5

Treatment of an Anemic Patient with ActRIIa-hFc

[0162]A clinical study was designed to treat patients with multiple doses
of ActRIIa-hFc, at dose levels of 0.1 mg/kg, 0.3 mg/kg and 1.0 mg/kg,
with dosing every thirty days. Normal healthy patients in the trial
exhibited an increase in hemoglobin and hematocrit that is consistent
with the increases seen in the Phase I clinical trial reported in Example
4, except that, in some instances, the hemoglobin and hematocrit were
elevated beyond the normal range. An anemic patient with hemoglobin of
approximately 7.5 also received two doses at the 1 mg/kg level, resulting
in a hemoglobin level of approximately 10.5 after two months. The
patient's anemia was a microcytic anemia, thought to be caused by chronic
iron deficiency.

[0163]In this study the effects of the in vivo administration of
ActRIIa-mFc on the frequency of hematopoietic progenitors in bone marrow
and spleen was analyzed. One group of mice was injected with PBS as a
control and a second group of mice administered two doses of ActRIIa-mFc
at 10 mg/kg and both groups sacrificed after 8 days. Peripheral blood was
used to perform complete blood counts and femurs and spleens were used to
perform in vitro clonogenic assays to assess the lymphoid, erythroid and
myeloid progenitor cell content in each organ. In the peripheral blood a
significant increase in the red blood cell and hemoglobin content was
seen in compound treated mice. In the femurs there was no difference in
the nucleated cell numbers or progenitor content between the control and
treated groups. In the spleens, the compound treated group experienced a
statistically significant increase in the nucleated cell number before
red blood cell lysis and in the mature erythroid progenitor (CFU-E)
colony number per dish, frequency and total progenitor number per spleen.
In addition, and increase was seen in the number of myeloid (CFU-GM),
immature erythroid (BFU-E) and total progenitor number per spleen.

[0164]Animals:

[0165]Sixteen BDF1 female mice 6-8 weeks of age were used in the study.
Eight mice were injected subcutaneously with test compound ActRIIa-mFc at
days 1 and 3 at a dose of 10 mg/kg and eight mice were injected
subcutaneously with vehicle control, phosphate buffered saline (PBS), at
a volume of 100 μL per mouse. All mice were sacrificed 8 days after
first injection in accordance with the relevant Animal Care Guidelines.
Peripheral blood (PB) samples from individual animals were collected by
cardiac puncture and used for complete blood counts and differential
(CBC/Diff). Femurs and spleens were harvested from each mouse.

[0166]Tests Performed:

[0167]CBC/Diff Counts

[0168]PB from each mouse was collected via cardiac puncture and placed
into the appropriate microtainer tubes. Samples were sent to CLV for
analysis on a CellDyn 3500 counter.

[0169]Clonogenic Assays

[0170]Clonogenic progenitors of the myeloid, erythroid and lymphoid
lineages were assessed using the in vitro methylcellulose-based media
systems described below.

[0178]Mouse femurs and spleens were processed by standard protocols.
Briefly, bone marrow was obtained by flushing the femoral cavity with
Iscove's Modified Dulbecco's Media containing 2% fetal bovine serum (IMDM
2% FBS) using a 21 gauge needle and 1 cc syringe. Spleen cells were
obtained by crushing spleens through a 70 μM filter and rinsing the
filter with IMDM 2% FBS. Nucleated cell counts in 3% glacial acetic acid
were then performed on the single cells suspensions using a Neubauer
counting chamber so that the total cells per organ could be calculated.
To remove contaminating red blood cells, total spleen cells were then
diluted with 3 times the volume of ammonium chloride lysis buffer and
incubated on ice 10 minutes. The cells were then washed and resuspended
in

[0179]IMDM 2% FBS and a second cell count were performed to determine the
cell concentration of cells after lysis.

[0180]Cell stocks were made and added to each methylcellulose-based media
formulation to obtain the optimal plating concentrations for each tissue
in each media formulation. Bone marrow cells were plated at
1×105 cells per dish in MethoCult® 3334 to assess mature
erythroid progenitors, 2×105 cells per dish in MethoCult®
3630 to assess lymphoid progenitors and 3×104 cells per dish
in MethoCult® 3434 to assess immature erythroid and myeloid
progenitors. Spleen cells were plated at 4×105 cells per dish
in MethoCult® 3334 to assess mature erythroid progenitors,
4×105 cells per dish in MethoCult® 3630 to assess lymphoid
progenitors and 2×105 cells per dish in MethoCult® 3434 to
assess immature erythroid and myeloid progenitors. Cultures plated in
triplicate dishes were incubated at 37° C., 5% CO2 until colony
enumeration and evaluation was performed by trained personnel. Mature
erythroid progenitors were cultured for 2 days, lymphoid progenitors were
cultured for 7 days and mature erythroid and myeloid progenitors were
cultured for 12 days.

[0181]Analysis:

[0182]The mean±1 standard deviation was calculated for the triplicate
cultures of the clonogenic assays and for the control and treatment
groups for all data sets.

[0183]Frequency of colony forming cells (CFC) in each tissue was
calculated as follows:

[0189]Standard t-tests were performed to assess if there was a differences
in the mean number of cells or hematopoietic progenitors between the PBS
control mice and compound treated mice. Due to the potential subjectivity
of colony enumeration, a p value of less than 0.01 is deemed significant.
Mean values (±SD) for each group are shown in the tables below.

[0190]Treatment of mice with ActRIIa-mFc resulted in significant increases
in a number of hematopoietic parameters. In the peripheral blood a
significant increase in the red blood cell and hemoglobin content was
seen in compound treated mice. In the femurs there was no difference in
the nucleated cell numbers or progenitor content between the control and
treated groups. In the spleens, the compound treated group experienced a
statistically significant increase in the nucleated cell number before
red blood cell lysis and in the mature erythroid progenitor (CFU-E)
colony number per dish, frequency and total progenitor number per spleen.
In addition, an increase was seen in the number of myeloid (CFU-GM),
immature erythroid (BFU-E) and total progenitor number per spleen.

Example 7

Alternative ActRIIa-Fc Proteins

[0191]A variety of ActRIIa variants that may be used according to the
methods described herein are described in the International Patent
Application published as WO2006/012627 (see e.g., pp. 55-58),
incorporated herein by reference in its entirety. An alternative
construct may have a deletion of the C-terminal tail (the final 15 amino
acids of the extracellular domain of ActRIIa. The sequence for such a
construct is presented below (Fc portion underlined)(SEQ ID NO: 12):

[0192]Applicants investigated the effect of ActRIIA-mFc on
chemotherapy-induced anemia in mice. In the first of two studies,
6-week-old female C57BL/6 mice were treated with a single dose of
ActRIIA-mFc (10 mg/kg, s.c.) or vehicle (phosphate-buffered saline) 3
days before a single dose of the chemotherapeutic paclitaxel (20 mg/kg,
i.p.). Blood samples were collected before chemotherapy and then 3, 7,
and 14 days (n=6 per cohort per time point) after paclitaxel. ActRIIA-mFc
prevented the decline in hematocrit level otherwise observed after
paclitaxel (FIG. 15), and similar effects were observed for hemoglobin
concentration and RBC count. In a second study, 6-week-old female C57BL/6
mice were given a varying number of ActRIIA-mFc doses (10 mg/kg, s.c.),
or vehicle (PBS), beginning before paclitaxel (20 mg/kg single dose,
i.p.) and continuing at intervals of 3 or 4 days. Blood samples were
collected 3, 7, and 14 days (n=8 per cohort per time point) after
paclitaxel. At 14 days, ActRIIA-mFc treatment increased hematocrit level
progressively as a function of dose number (FIG. 16). Thus, ActRIIA-mFc
can stimulate erythropoiesis sufficiently to attenuate or prevent
chemotherapy-induced anemia.

Example 9

Effect of ActRIIA-mFc on Anemia in a Mouse Model of Chronic Kidney Disease

[0193]Applicants investigated the effect of ActRIIA-mFc on
nephrectomy-induced anemia in mice as a model of chronic kidney disease.
In the first of two studies, female C57BL/6 mice underwent a partial
surgical nephrectomy, with removal of approximately five-sixths of total
kidney volume, to reduce production of erythropoietin. Mice were given a
4-week recovery period with a high-fat diet to further promote renal
deficiency and were then treated twice-weekly with ActRIIA-mFc (10 mg/kg,
s.c.) or vehicle (PBS) for a total of 8 weeks. Blood samples were
collected before the onset of dosing, after 4 weeks of treatment, and
after 8 weeks of treatment (n=8 per cohort per time point). Control mice
exhibited a decline in hematocrit level over the 8-week treatment period,
whereas ActRIIA-mFc treatment prevented the decline at 4 weeks and also
produced a beneficial trend at 8 weeks (FIG. 17). Similar benefits of
ActRIIA-mFc treatment over control were observed in a second study that
differed mainly in the use of a longer recovery period (2 months) and a
standard diet. Thus, ActRIIA-mFc can stimulate erythropoiesis
sufficiently to prevent or attenuate anemia in a model of chronic kidney
disease.

[0194]Taken together, these findings indicate that soluble ActRIIA-Fc
fusion proteins can be used as antagonists of signaling by TGF-family
ligands to increase circulating levels of red blood cells, and thereby,
to treat hypoproliferative anemias resulting from chronic diseases such
as cancer and renal disease, and potentially other inflammatory or
infectious diseases as well. Note that effects of ACE-011 on anemia in
human patients are typically robust compared to the more modest effects
in rodents.

INCORPORATION BY REFERENCE

[0195]All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated to be
incorporated by reference.

[0196]While specific embodiments of the subject matter have been
discussed, the above specification is illustrative and not restrictive.
Many variations will become apparent to those skilled in the art upon
review of this specification and the claims below. The full scope of the
invention should be determined by reference to the claims, along with
their full scope of equivalents, and the specification, along with such
variations.